Flea allantoinase nucleic acid molecules, proteins and uses thereof

ABSTRACT

The present invention relates to flea allantoinase proteins; to flea allantoinase nucleic acid molecules, including those that encode such flea allantoinase proteins; to antibodies raised against such proteins; and to compounds that inhibit the activity of such proteins. The present invention also includes methods to obtain such proteins, nucleic acid molecules, antibodies, and inhibitory compounds. The present invention also includes therapeutic compositions comprising such inhibitory compounds, particularly those that specifically inhibit flea allantoinase activity, as well as the use of such therapeutic compositions to treat animals.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/543,668, filed Apr. 7, 2000 entitled “FLEA ALLANTOINASENUCLEIC ACID MOLECULES, PROTEINS AND USES THEREOF, now abandoned, andclaims priority to U.S. Provisional Patent Application Serial No.60/128,704, filed Apr. 9, 1999 entitled “NOVEL FLEA HEAD, NERVE CORD,NERVE CORD, HINDGUT AND MALPIGHIAN TUBULE NUCLEIC ACID MOLECULES,PROTEINS AND USES THEREOF”.

FIELD OF THE INVENTION

The present invention relates to flea allantoinase nucleic acidmolecules, proteins encoded by such nucleic acid molecules, antibodiesraised against such proteins, inhibitors of such proteins and methods todetect such inhibitors. The present invention also includes therapeuticcompositions comprising such nucleic acid molecules, proteins,antibodies, and/or other inhibitors, as well as uses thereof.

BACKGROUND OF THE INVENTION

Flea infestation of animals is a health and economic concern becausefleas arc known to cause and/or transmit a variety of diseases. Fleasdirectly cause a variety of diseases, including allergies, and alsocarry a variety of infectious agents including, but not limited to,endoparasites (e.g., nematodes, cestodes, trematodes and protozoa),bacteria and viruses. In particular, the bites of fleas are a problemfor animals maintained as pets because the infestation becomes a sourceof annoyance not only for the pet but also for the pet owner who mayfind his or her home generally contaminated with insects. As such, fleasare a problem not only when they are on an animal but also when they arein the general environment of the animal.

Bites from fleas are a particular problem because they not only can leadto disease transmission but also can cause a hypersensitive response inanimals which is manifested as disease. For example, bites from fleascan cause an allergic disease called flea allergic (or allergy)dermatitis (FAD). A hypersensitive response in animals typically resultsin localized tissue inflammation and damage, causing substantialdiscomfort to the animal.

The medical importance of flea infestation has prompted the developmentof reagents capable of controlling flea infestation. Commonlyencountered methods to control flea infestation are generally focused onuse of insecticides. While some of these products are efficacious, most,at best, offer protection of a very limited duration. Furthermore, manyof the methods are often not successful in reducing flea populations. Inparticular, insecticides have been used to prevent flea infestation ofanimals by adding such insecticides to shampoos, powders, collars,sprays, spot-on formulations foggers and liquid bath treatments (i.e.,dips). Reduction of flea infestation on the pet has been unsuccessfulfor one or more of the following reasons: failure of owner compliance(frequent administration is required); behavioral or physiologicalintolerance of the pet to the pesticide product or means ofadministration; and the emergence of flea populations resistant to theprescribed dose of pesticide.

Allantoinase is involved in the catalysis of the reaction convertingallantoin to allantoic acid. This is a middle step in purine catabolism,which in insects results in the secretion of urea as the end product.The enzyme is located in the peroxisomes of the liver and kidney inamphibians. There is no known mammalian homologue to allantoinase, asmammals secrete uric acid, a precursor to allantoin. As such, fleaallantoinase represents a novel target for anti-flea vaccines andchemotherapeutic drugs. Therefore, isolation and sequencing of fleaallantoinase genes may be critical for use in identifying specificagents for treating animals for flea infestation.

SUMMARY OF THE INVENTION

The present invention relates to a novel product and process forprotection of animals from flea infestation.

The present invention provides flea allantoinase proteins; nucleic acidmolecules encoding flea allantoinase proteins; antibodies raised againstsuch proteins; mimetopes of such proteins or antibodies; and compoundsthat inhibit flea allantoinase activity (i.e, inhibitory compounds orinhibitors).

The present invention also includes methods to obtain such proteins,mimetopes, nucleic acid molecules, antibodies and inhibitory compounds.The present invention also includes the use of proteins and antibodiesto identify such inhibitory compounds as well as assay kits to identifysuch inhibitory compounds. Also included in the present invention aretherapeutic compositions comprising proteins, mimetopes, nucleic acidmolecules, antibodies and inhibitory compounds of the present inventionincluding protective compounds derived from a protein of the presentinvention that inhibit the activity of allantoinase proteins; alsoincluded are uses of such therapeutic compounds to reduce fleainfestation.

One embodiment of the present invention is an isolated nucleic acidmolecule that hybridizes with a nucleic acid sequence having SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:9, and SEQ ID NO:11 under conditions that allow less than orequal to about 30% base pair mismatch.

Another embodiment of the present invention is an isolated nucleic acidmolecule having nucleic acid sequence that is at least about 70%identical to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:11.

The present invention also relates to recombinant molecules, recombinantviruses and recombinant cells that include a nucleic acid molecule ofthe present invention. Also included are methods to produce such nucleicacid molecules, recombinant molecules, recombinant viruses andrecombinant cells.

Another embodiment of the present invention includes an isolated proteinthat is at least about 70% identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NO:2, SEQ ID NO:7 and SEQ ID NO:10and fragments thereof, wherein such fragments can elicit an immuneresponse against respective flea proteins or have activity comparable torespective flea proteins.

Another embodiment of the present invention includes an isolated proteinencoded by a nucleic acid molecule that hybridizes with the complementof a nucleic acid sequence having SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6and SEQ ID NO:9, under conditions that allow less than or equal to about30% base pair mismatch.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for flea allantoinase nucleic acidmolecules, proteins encoded by such nucleic acid molecules, antibodiesraised against such proteins, and inhibitors of such proteins. As usedherein, flea allantoinase nucleic acid molecules and proteins encoded bysuch nucleic acid molecules are also referred to as allantoinase nucleicacid molecules and proteins. Flea allantoinase nucleic acid moleculesand proteins of the present invention can be isolated from a flea orprepared recombinantly or synthetically. Flea allantoinase nucleic acidmolecules of the present invention can be RNA or DNA, or modified formsthereof, and can be double-stranded or single-stranded; examples ofnucleic acid molecules include, but are not limited to, complementaryDNA (cDNA) molecules, genomic DNA molecules, synthetic DNA molecules,DNA molecules which are specific tags for messenger RNA, andcorresponding mRNA molecules. As such, a flea nucleic acid molecule ofthe present invention is not intended refer to an entire chromosomewithin which such a nucleic acid molecule is contained, however, a fleaallantoinase cDNA of the present invention may include all regions suchas regulatory regions that control production of flea allantoinaseproteins encoded by such a cDNA (such as, but not limited to,transcription, translation or post-translation control regions) as wellas the coding region itself, and any introns or non-translated codingregions. As used herein, the phrase “flea allantoinase protein” refersto a protein encoded by a flea allantoinase nucleic acid molecule.

Flea allantoinase nucleic acid molecules of known length isolated from aflea, such as Ctenocephalides felis are denoted “nCfALN₁₉₀ ”, forexample nCfALN₂₀₃₅, wherein “#” refers to the number of nucleotides inthat molecule, and flea allantoinase proteins of known length aredenoted “PCfALN_(#)” (for example PCfALN₄₈₃) wherein “#” refers to thenumber of amino acid residues in that molecule.

The present invention also provides for flea allantoinase DNA moleculesthat are specific tags for messenger RNA molecules. Such DNA moleculescan correspond to an entire or partial sequence of a messenger RNA, andtherefore, a DNA molecule corresponding to such a messenger RNA molecule(i.e. a cDNA molecule), can encode a full-length or partial-lengthprotein. A nucleic acid molecule encoding a partial-length protein canbe used directly as a probe or indirectly to generate primers toidentify and/or isolate a cDNA nucleic acid molecule encoding acorresponding, or structurally related, full-length protein. Such apartial cDNA nucleic acid molecule can also be used in a similar mannerto identify a genomic nucleic acid molecule, such as a nucleic acidmolecule that contains the complete gene including regulatory regions,exons and introns. Methods for using partial flea allantoinase cDNAmolecules and sequences to isolate full-length and corresponding cDNAmolecules are described in the examples herein below.

The proteins and nucleic acid molecules of the present invention can beobtained from their natural source, or can be produced using, forexample, recombinant nucleic acid technology or chemical synthesis. Alsoincluded in the present invention is the use of these proteins andnucleic acid molecules as well as antibodies and inhibitory compoundsthereto as therapeutic compositions to protect animals from fleainfestation, as well as in other applications, such as those disclosedbelow.

One embodiment of the present invention is an isolated protein thatincludes a flea allantoinase protein. It is to be noted that the term“a” or “an” entity refers to one or more of that entity; for example, aprotein, a nucleic acid molecule, an antibody and a therapeuticcomposition refers to “one or more” or “at least one” protein, nucleicacid molecule, antibody and therapeutic composition respectively. Assuch, the terms “a” (or “an”), “one or more” and “at least one” can beused interchangeably herein. It is also to be noted that the terms“comprising”, “including”, and “having” can be used interchangeably.According to the present invention, an isolated, or biologically pure,flea allantoinase protein, is a protein that has been removed from itsnatural milieu, such as a flea protein extract having allantoinaseactivity. As such, “isolated” and “biologically pure” do not necessarilyreflect the extent to which the protein has been purified. An isolatedprotein of the present invention can be obtained from its naturalsource, can be produced using recombinant DNA technology, or can beproduced by chemical synthesis.

As used herein, isolated flea allantoinase proteins of the presentinvention can be full-length proteins or any homologue of such proteins.An isolated protein of the present invention, including a homologue, canbe identified in a straight-forward manner by the protein's ability toelicit an immune response against a flea allantoinase protein or by theprotein's ability to exhibit flea allantoinase activity. Examples offlea allantoinase homologue proteins include flea allantoinase proteinsin which amino acids have been deleted (e.g., a truncated version of theprotein, such as a peptide), inserted, inverted, substituted and/orderivatized (e.g., by glycosylation, phosphorylation, acetylation,myristoylation, prenylation, palmitoylation, amidation and/or additionof glycerophosphatidyl inositol) such that the homologue includes atleast one epitope capable of eliciting an immune response against a fleaallantoinase protein, and/or of binding to an antibody directed againsta flea allantoinase protein. That is, when the homologue is administeredto an animal as an immunogen, using techniques known to those skilled inthe art, the animal will produce an immune response against at least oneepitope of a natural flea allantoinase protein. The ability of a proteinto effect an immune response can be measured using techniques known tothose skilled in the art. As used herein, the term “epitope” refers tothe smallest portion of a protein or other antigen capable ofselectively binding to the antigen binding site of an antibody or a Tcell receptor. It is well accepted by those skilled in the art that theminimal size of a protein epitope is about four to six amino acids. Asis appreciated by those skilled in the art, an epitope can include aminoacids that naturally are contiguous to each other as well as amino acidsthat, due to the tertiary structure of the natural protein, are insufficiently close proximity to form an epitope. According to thepresent invention, an epitope includes a portion of a protein comprisingat least 4 amino acids, at least 5 amino acids, at least 6 amino acids,at least 10 amino acids, at least 15 amino acids, at least 20 aminoacids, at least 25 amino acids, at least 30 amino acids, at least 35amino acids, at least 40 amino acids or at least 50 amino acids inlength.

In one embodiment of the present invention a flea allantoinase homologueprotein has flea allantoinase activity, i.e. the homologue exhibits anactivity similar to its natural counterpart. Methods to detect andmeasure such activities are known to those skilled in the art.

Flea allantoinase homologue proteins can be the result of naturalallelic variation or natural mutation. Flea allantoinase proteinhomologues of the present invention can also be produced usingtechniques known in the art including, but not limited to, directmodifications to the protein or modifications to the gene encoding theprotein using, for example, classic or recombinant DNA techniques toeffect random or targeted mutagenesis.

Flea allantoinase proteins of the present invention are encoded by fleaallantoinase nucleic acid molecules. As used herein, flea allantoinasenucleic acid molecules include nucleic acid sequences related to naturalflea allantoinase genes, and, preferably, to C. felis flea allantoinasegenes. As used herein, flea allantoinase genes include all regions suchas regulatory regions that control production of flea allantoinaseproteins encoded by such genes (such as, but not limited to,transcription, translation or post-translation control regions) as wellas the coding region itself, and any introns or non-translated codingregions. As used herein, a nucleic acid molecule that “includes” or“comprises” a sequence may include that sequence in one contiguousarray, or may include the sequence as fragmented exons such as is oftenfound for a flea gene. As used herein, the term “coding region” refersto a continuous linear array of nucleotides that translates into aprotein. A full-length coding region is that coding region that istranslated into a full-length, i.e., a complete protein as would beinitially translated in its natural millieu, prior to anypost-translational modifications.

One embodiment of the present invention is a C. felis flea allantoinasegene that includes the nucleic acid sequence SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, and SEQID NO:11. These nucleic acid sequences are further described herein. Forexample, nucleic acid sequence SEQ ID NO:1 represents the deducedsequence of the coding strand of a C. felis cDNA denoted herein as C.felis allantoinase nucleic acid molecule nCfALN₂₀₃₅, the production ofwhich is disclosed in the Examples. Nucleic acid molecule SEQ ID NO:1comprises an apparently full-length coding region. The complement of SEQID NO:1 (represented herein by SEQ ID NO:3) refers to the nucleic acidsequence of the strand fully complementary to the strand having SEQ IDNO:1, which can easily be determined by those skilled in the art.Likewise, a nucleic acid sequence complement of any nucleic acidsequence of the present invention refers to the nucleic acid sequence ofthe nucleic acid strand that is fully complementary to (i.e., can form acomplete double helix with) the strand for which the sequence is cited.It should be noted that since nucleic acid sequencing technology is notentirely error-free, SEQ ID NO:1 (as well as other nucleic acid andprotein sequences presented herein) represents an apparent nucleic acidsequence of the nucleic acid molecule encoding a flea allantoinaseprotein of the present invention.

Translation of SEQ ID NO:1, the coding strand of nCfALN₂₀₃₅, as well astranslation of SEQ ID NO:4, the coding strand of nCfALN₁₄₄₉, whichrepresents the coding region of SEQ ID NO:1, each yields a protein ofabout 483 amino acids, denoted herein as PCfALN₄₈₃, the amino acidsequence of which is presented in SEQ ID NO:2, assuming a first in-framecodon extending from nucleotide 1 to nucleotide 3 of SEQ ID NO:4.

In one embodiment, a gene or other nucleic acid molecule of the presentinvention can be an allelic variant that includes a similar but notidentical sequence to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:11. Forexample, an allelic variant of a C. felis allantoinase gene includingSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:9, and SEQ ID NO:11 is a gene that occurs at essentiallythe same locus (or loci) in the genome as the gene including SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:9, and SEQ ID NO:11, but which, due to natural variationscaused by, for example, mutation or recombination, has a similar but notidentical sequence. Because natural selection typically selects againstalterations that affect function, allelic variants (i.e. allelescorresponding to, or of, cited nucleic acid sequences) usually encodeproteins having similar activity to that of the protein encoded by thegene to which they are being compared. Allelic variants of genes ornucleic acid molecules can also comprise alterations in the 5′ or 3′untranslated regions of the gene (e.g., in regulatory control regions),or can involve alternative splicing of a nascent transcript, therebybringing alternative exons into juxtaposition. Allelic variants are wellknown to those skilled in the art and would be expected to occurnaturally within a given flea species, since the genome is diploid, andsexual reproduction will result in the reassortment of alleles.

In one embodiment of the present invention, isolated flea allantoinaseproteins are encoded by nucleic acid molecules that hybridize understringent hybridization conditions to genes or other nucleic acidmolecules encoding flea allantoinase proteins, respectively. The minimalsize of flea allantoinase proteins of the present invention is a sizesufficient to be encoded by a nucleic acid molecule capable of forming astable hybrid (i.e., hybridizing under stringent hybridizationconditions) with the complementary sequence of a nucleic acid moleculeencoding the corresponding natural protein. The size of a nucleic acidmolecule encoding such a protein is dependent on the nucleic acidcomposition and the percent homology between the flea allantoinasenucleic acid molecule and the complementary nucleic acid sequence. Itcan easily be understood that the extent of homology required to form astable hybrid under stringent conditions can vary depending on whetherthe homologous sequences are interspersed throughout a given nucleicacid molecule or are clustered (i.e., localized) in distinct regions ona given nucleic acid molecule.

The minimal size of a nucleic acid molecule capable of forming a stablehybrid with a gene encoding a flea allantoinase protein is at leastabout 12 to about 15 nucleotides in length if the nucleic acid moleculeis GC-rich and at least about 15 to about 17 bases in length if it isAT-rich. The minimal size of a nucleic acid molecule used to encode aflea allantoinase protein homologue of the present invention is fromabout 12 to about 18 nucleotides in length. Thus, the minimal size offlea allantoinase protein homologues of the present invention is fromabout 4 to about 6 amino acids in length. There is no limit, other thana practical limit, on the maximal size of a nucleic acid moleculeencoding a flea allantoinase protein of the present invention because anucleic acid molecule of the present invention can include a portion ofa gene or cDNA or RNA, an entire gene or cDNA or RNA, or multiple genesor cDNA or RNA. The preferred size of a protein encoded by a nucleicacid molecule of the present invention depends on whether a full-length,fusion, multivalent, or functional portion of such a protein is desired.

Stringent hybridization conditions are determined based on definedphysical properties of the flea allantoinase nucleic acid molecule towhich the nucleic acid molecule is being hybridized, and can be definedmathematically. Stringent hybridization conditions are thoseexperimental parameters that allow an individual skilled in the art toidentify significant similarities between heterologous nucleic acidmolecules. These conditions are well known to those skilled in the art.See, for example, Sambrook, et al., 1989, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Labs Press, and Meinkoth, et al.,1984, Anal. Biochem. 138, 267-284, each of which is incorporated byreference herein in its entirety. As explained in detail in the citedreferences, the determination of hybridization conditions involves themanipulation of a set of variables including the ionic strength (M, inmoles/liter), the hybridization temperature (° C.), the concentration ofnucleic acid helix destabilizing agents (such as formamide), the averagelength of the shortest hybrid duplex (n), and the percent G+Ccomposition of the fragment to which an unknown nucleic acid molecule isbeing hybridized. For nucleic acid molecules of at least about 150nucleotides, these variables are inserted into a standard mathematicalformula to calculate the melting temperature, or T_(m), of a givennucleic acid molecule. As defined in the formula below, T_(m) is thetemperature at which two complementary nucleic acid molecule strandswill disassociate, assuming 100% complementarity between the twostrands:

T_(m)=81.5° C.+16.6 log M+0.41(% G+C)−500/n−0.61(% formamide).

For nucleic acid molecules smaller than about 50 nucleotides, hybridstability is defined by the dissociation temperature (T_(d)), which isdefined as the temperature at which 50% of the duplexes dissociate. Forthese smaller molecules, the stability at a standard ionic strength isdefined by the following equation:

 T_(d)=4(G+C)+2(A+T).

A temperature of 5° C. below T_(d) is used to detect hybridizationbetween perfectly matched molecules.

Also well known to those skilled in the art is how base pair mismatch,i.e. differences between two nucleic acid molecules being compared,including non-complementarity of bases at a given location, and gaps dueto insertion or deletion of one or more bases at a given location oneither of the nucleic acid molecules being compared, will affect T_(m)or T_(d) for nucleic acid molecules of different sizes. For example,T_(m) decreases about 1° C. for each 1% of mismatched base pairs forhybrids greater than about 150 bp, and T_(d) decreases about 5° C. foreach mismatched base pair for hybrids below about 50 bp. Conditions forhybrids between about 50 and about 150 base pairs can be determinedempirically and without undue experimentation using standard laboratoryprocedures well known to those skilled in the art. These simpleprocedures allow one skilled in the art to set the hybridizationconditions (by altering, for example, the salt concentration, theconcentration of helix destabilizing agents, or the temperature) so thatonly nucleic acid hybrids with greater than a specified % base pairmismatch will hybridize. Because one skilled in the art can easilydetermine whether a given nucleic acid molecule to be tested is lessthan or greater than about 50 nucleotides, and can therefore choose theappropriate formula for determining hybridization conditions, he or shecan determine whether the nucleic acid molecule will hybridize with agiven gene under conditions designed to allow a desired amount of basepair mismatch.

Hybridization reactions are often carried out by attaching the nucleicacid molecule to be hybridized to a solid support such as a membrane,and then hybridizing with a labeled nucleic acid molecule, typicallyreferred to as a probe, suspended in a hybridization solution. Examplesof common hybridization reaction techniques include, but are not limitedto, the well-known Southern and northern blotting procedures. Typically,the actual hybridization reaction is done under non-stringentconditions, i.e., at a lower temperature and/or a higher saltconcentration, and then high stringency is achieved by washing themembrane in a solution with a higher temperature and/or lower saltconcentration in order to achieve the desired stringency.

For example, if the skilled artisan wished to identify a nucleic acidmolecule that hybridizes under conditions that would allow less than orequal to 30% pair mismatch with a flea allantoinase nucleic acidmolecule of about 150 bp in length or greater, the following conditionscould preferably be used. The average G+C content of flea DNA is about37%, as calculated from known flea nucleic acid sequences. The unknownnucleic acid molecules would be attached to a support membrane, and the150 bp probe would be labeled, e.g. with a radioactive tag. Thehybridization reaction could be carried out in a solution comprising2×SSC in the absence of nucleic acid helix destabilizing compounds, at atemperature of about 37° C. (low stringency conditions). Solutions ofdiffering concentrations of SSC can be made by one of skill in the artby diluting a stock solution of 20×SSC (175.3 gram NaCl and about 88.2gram sodium citrate in 1 liter of water, pH 7) to obtain the desiredconcentration of SSC. The skilled artisan would calculate the washingconditions required to allow up to 30% base pair mismatch. For example,in a wash solution comprising 1×SSC in the absence of nucleic acid helixdestabilizing compounds, the T_(m) of perfect hybrids would be about 77°C.:

81.5° C.+16.6 log (0.15M)+(0.41×73)−(500/150)−(0.61×0)=77.5° C.

Thus, to achieve hybridization with nucleic acid molecules having about30% base pair mismatch, hybridization washes would be carried out at atemperature of less than or equal to 47.5° C. It is thus within theskill of one in the art to calculate additional hybridizationtemperatures based on the desired percentage base pair mismatch,formulae and G/C content disclosed herein. For example, it isappreciated by one skilled in the art that as the nucleic acid moleculeto be tested for hybridization against nucleic acid molecules of thepresent invention having sequences specified herein becomes longer than150 nucleotides, the T_(m) for a hybridization reaction allowing up to30% base pair mismatch will not vary significantly from 47.5° C.

Furthermore, it is known in the art that there are commerciallyavailable computer programs for determining the degree of similaritybetween two nucleic acid or protein sequences. These computer programsinclude various known methods to determine the percentage identity andthe number and length of gaps between hybrid nucleic acid molecules orproteins. Preferred methods to determine the percent identity amongamino acid sequences and also among nucleic acid sequences includeanalysis using one or more of the commercially available computerprograms designed to compare and analyze nucleic acid or amino acidsequences. These computer programs include, but are not limited to, theSeqLab® Wisconsin Package™ Version 10.0-UNIX sequence analysis software,available from Genetics Computer Group, Madison, Wis. (hereinafter“SeqLab”); and DNAsis® sequence analysis software, version 2.0,available from Hitachi Software, San Bruno, Calif. (hereinafter“DNAsis”). Such software programs represent a collection of algorithmspaired with a graphical user interface for using the algorithms. TheDNAsis and SeqLab software, for example, employ a particular algorithm,the Needleman-Wunsch algorithm to perform pair-wise comparisons betweentwo sequences to yield a percentage identity score, see Needleman, S. B.and Wunsch, C. D., 1970, J. Mol. Biol., 48, 443, which is incorporatedherein by reference in its entirety. Such algorithms, including theNeedleman-Wunsch algorithm, are commonly used by those skilled in thenucleic acid and amino acid sequencing art to compare sequences. Apreferred method to determine percent identity among amino acidsequences and also among nucleic acid sequences includes using theNeedleman-Wunsch algorithm, available in the SeqLab software, using thePairwise Comparison/Gap function with the nwsgapdna.cmp scoring matrix,the gap creation penalty and the gap extension penalties set at defaultvalues, and the gap shift limits set at maximum (hereinafter referred toas “SeqLab default parameters”). An additional preferred method todetermine percent identity among amino acid sequences and also amongnucleic acid sequences includes using the Higgins-Sharp algorithm,available in the DNAsis software, with the gap penalty set at 5, thenumber of top diagonals set at 5, the fixed gap penalty set at 10, thek-tuple set at 2, the window size set at 5, and the floating gap penaltyset at 10. A particularly preferred method to determine percent identityamong amino acid sequences and also among nucleic acid sequencesincludes using the Needleman-Wunsch algorithm available in the SeqLabsoftware, using the SeqLab default parameters.

One embodiment of the present invention includes a flea allantoinaseprotein. A preferred flea allantoinase protein includes a proteinencoded by a nucleic acid molecule that hybridizes under conditions thatpreferably allow less than or equal to 30% base pair mismatch,preferably under conditions that allow less than or equal to 20% basepair mismatch, preferably under conditions that allow less than or equalto 10% base pair mismatch, preferably under conditions that allow lessthan or equal to 8% base pair mismatch, preferably under conditions thatallow less than or equal to 5% base pair mismatch or preferably underconditions that allow less than or equal to 2% base pair mismatch with anucleic acid molecule selected from the group consisting of SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:8 and SEQ ID NO:11.

Another embodiment of the present invention includes a flea allantoinaseprotein encoded by a nucleic acid molecule that hybridizes underconditions comprising, (a) hybridizing in a solution comprising 1×SSC inthe absence of nucleic acid helix destabilizing compounds, at atemperature of 37° C. and (b) washing in a solution comprising 1×SSC inthe absence of nucleic acid helix destabilizing compounds, at atemperature of 47° C., to an isolated nucleic acid molecule selectedfrom the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8 andSEQ ID NO:11.

Another preferred flea allantoinase protein of the present inventionincludes a protein that is encoded by a nucleic acid molecule that ispreferably at least 70% identical, preferably at least 80% identical,preferably at least 90% identical, preferably at least 92% identical,preferably at least 95% identical or preferably at least 98% identicalto a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:1,SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:9; also preferred are fragments(i.e. portions) of such proteins encoded by nucleic acid molecules thatare at least 30 nucleotides. Percent identity as used herein isdetermined using the Needleman-Wunsch algorithm, available in the SeqLabsoftware using default parameters.

Additional preferred flea allantoinase proteins of the present inventioninclude proteins having the amino acid sequence SEQ ID NO:2, SEQ ID NO:7and SEQ ID NO:10, and proteins comprising homologues of a protein havingthe amino acid sequence SEQ ID NO:2, SEQ ID NO:7 and SEQ ID NO:10,wherein such a homologue comprises at least one epitope that elicits animmune response against a protein having an amino acid sequence SEQ IDNO:2, SEQ ID NO:7 and SEQ ID NO:10. Likewise, also preferred areproteins encoded by nucleic acid molecules comprising nucleic acidsequence SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:9, or byhomologues thereof.

A preferred isolated flea allantoinase protein of the present inventionis a protein encoded by at least one of the following nucleic acidmolecules: nCfALN₂₀₃₅, nCfALN₁₄₄₉, nCfALN₁₃₈₃, and nCfALN₁₁₂₃, orallelic variants of any of these nucleic acid molecules. Also preferredis an isolated protein encoded by a nucleic acid molecule having nucleicacid sequence SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:9; ora protein encoded by an allelic variant of any of these listed nucleicacid molecules.

Preferred flea allantoinase proteins of the present invention includeproteins having amino acid sequences that are at least 70%, preferably80%, preferably 90%, preferably 92%, preferably 95%, preferably at least98%, preferably at least 99%, or preferably 100% identical to amino acidsequence SEQ ID NO:2, SEQ ID NO:7 and SEQ ID NO:10 and proteins encodedby allelic variants of nucleic acid molecules encoding flea allantoinaseproteins having amino acid sequences SEQ ID NO:2, SEQ ID NO:7 and SEQ IDNO:10. Also preferred are fragments thereof having at least 10 aminoacid residues.

In one embodiment of the present invention, C. felis allantoinaseproteins comprise amino acid sequence SEQ ID NO:2, SEQ ID NO:7 and SEQID NO:10 (including, but not limited to, the proteins consisting ofamino acid sequence SEQ ID NO:2, SEQ ID NO:7 and SEQ ID NO:10, fusionproteins and multivalent proteins), and proteins encoded by allelicvariants of nucleic acid molecules encoding proteins having amino acidsequence SEQ ID NO:2, SEQ ID NO:7 and SEQ ID NO:10.

In one embodiment, a preferred flea allantoinase protein comprises anamino acid sequence of at least 6 amino acids, preferably at least 10amino acids, preferably at least 15 amino acids, preferably at least 20amino acids, preferably at least 25 amino acids, preferably at least 30amino acids, preferably at least 35 amino acids, preferably at least 40amino acids, preferably at least 50 amino acids, preferably at least 75amino acids, preferably at least 100 amino acids, preferably at least125 amino acids, preferably at least 150 amino acids, preferably atleast 175 amino acids, preferably at least 200 amino acids, preferablyat least 250 amino acids, preferably at least 300 amino acids,preferably at least 350 amino acids, preferably at least 400 aminoacids, preferably at least 450 amino acids, preferably at least 475amino acids, or preferably at least 480 amino acids. In anotherembodiment, preferred flea allantoinase proteins comprise full-lengthproteins, i.e., proteins encoded by full-length coding regions, orpost-translationally modified proteins thereof, such as mature proteinsfrom which initiating methionine and/or signal sequences or “pro”sequences have been removed.

Additional preferred flea allantoinase proteins of the present inventioninclude proteins encoded by nucleic acid molecules comprising at least aportion of nCfALN_(2 ±), nCfALN₁₄₄₉, nCfALN₁₃₈₃, and nCfALN₁₁₂₃, as wellas flea allantoinase proteins encoded by allelic variants of suchnucleic acid molecules. A portion of such flea allantoinase nucleic acidmolecule is preferably at least 30 nucleotides in length.

Also preferred are flea allantoinase proteins encoded by nucleic acidmolecules having nucleic acid sequences comprising at least a portion ofSEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:9, as well asallelic variants of these nucleic acid molecules. A portion of such fleaallantoinase nucleic acid molecule is preferably at least 30 nucleotidesin length.

In another embodiment, a preferred flea allantoinase protein of thepresent invention is encoded by a nucleic acid molecule comprising atleast 20 nucleotides, preferably at least 25 nucleotides, preferably atleast 30 nucleotides, preferably at least 40 nucleotides, preferably atleast 50 nucleotides, preferably at least 75 nucleotides, preferably atleast 100 nucleotides, preferably at least 200 nucleotides, preferablyat least 400 nucleotides, preferably at least 500 nucleotides,preferably at least 750 nucleotides, preferably at least 1000nucleotides, preferably at least 1500 nucleotides, preferably at least1800 nucleotides, preferably at least 2000 nucleotides, or preferably atleast 2035 nucleotides. Within this embodiment is a flea allantoinaseprotein encoded by at least a portion of nCfALN₂₀₃₅, nCfALN₁₄₄₉,nCfALN₁₃₈₃, and nCfALN₁₁₂₃, or by an allelic variant of any of thesenucleic acid molecules. Preferred flea allantoinase proteins of thepresent invention are encoded by nucleic acid molecules comprisingapparently full-length flea allantoinase coding region, i.e., nucleicacid molecules encoding an apparently full-length flea allantoinaseprotein.

Preferred flea allantoinase proteins of the present invention can beused to develop inhibitors that, when administered to an animal in aneffective manner, are capable of protecting that animal from fleainfestation. In accordance with the present invention, the ability of aninhibitor of the present invention to protect an animal from fleainfestation refers to the ability of that protein to, for example,treat, ameliorate and/or prevent infestation caused by fleas. Inparticular, the phrase “to protect an animal from flea infestation”refers to reducing the potential for flea population expansion on andaround the animal (i.e., reducing the flea burden). Preferably, the fleapopulation size is decreased, optimally to an extent that the animal isno longer bothered by fleas. A host animal, as used herein, is an animalfrom which fleas can feed by attaching to and feeding through the skinof the animal. Fleas, and other ectoparasites, can live on a host animalfor an extended period of time or can attach temporarily to an animal inorder to feed. At any given time, a certain percentage of a fleapopulation can be on a host animal whereas the remainder can be in theenvironment of the animal. Such an environment can include not onlyadult fleas, but also flea eggs and/or flea larvae. The environment canbe of any size such that fleas in the environment are able to jump ontoand off of a host animal. For example, the environment of an animal caninclude plants, such as crops, from which fleas infest an animal. Assuch, it is desirable not only to reduce the flea burden on an animalper se, but also to reduce the flea burden in the environment of theanimal.

Suitable fleas to target include any flea that is essentially incapableof causing disease in an animal administered an inhibitor of the presentinvention. As such, fleas to target include any flea that produces aprotein that can be targeted by an inhibitory compound that inhibits aflea allantoinase protein function, thereby resulting in the decreasedability of the parasite to cause disease in an animal. Preferred fleasto target include fleas of the following genera: Ctenocephalides,Cyopsyllus, Diamanus (Oropsylla), Echidnophaga, Nosopsyllus, Pulex,Tunga, and Xenopsylla, with those of the species Ctenocephalides canis,Ctenocephalides felis, Diamanus montanus, Echidnophaga gallinacea,Nosopsyllus faciatus, Pulex irritans, Pulex simulans, Tunga penetransand Xenopsylla cheopis being more preferred, with C. felis being evenmore preferred. Such fleas are also preferred for the isolation ofproteins or nucleic acid molecules of the present invention.

One embodiment of a flea allantoinase protein of the present inventionis a fusion protein that includes a flea allantoinase protein-containingdomain attached to one or more fusion segments. Suitable fusion segmentsfor use with the present invention include, but are not limited to,segments that can: enhance a protein's stability; act as animmunopotentiator; and/or assist in purification of a flea allantoinaseprotein (e.g., by affinity chromatography). A suitable fusion segmentcan be a domain of any size that has the desired function (e.g., impartsincreased stability, imparts increased immunogenicity to a protein,and/or simplifies purification of a protein). Fusion segments can bejoined to amino and/or carboxyl termini of the fleaallantoinase-containing domain of the protein and can be susceptible tocleavage in order to enable straight-forward recovery of a fleaallantoinase protein. Fusion proteins are preferably produced byculturing a recombinant cell transformed with a fusion nucleic acidmolecule that encodes a protein including the fusion segment attached toeither the carboxyl and/or amino terminal end of a fleaallantoinase-containing domain. Preferred fusion segments include ametal binding domain (e.g., a poly-histidine segment); an immunoglobulinbinding domain (e.g., Protein A; Protein G; T cell; B cell; Fc receptoror complement protein antibody-binding domains); a sugar binding domain(e.g., a maltose binding domain); and/or a “tag” domain (e.g., at leasta portion of β-galactosidase, a strep tag peptide, a T7 tag peptide, aFlag™ peptide, or other domains that can be purified using compoundsthat bind to the domain, such as monoclonal antibodies). More preferredfusion segments include metal binding domains, such as a poly-histidinesegment; a maltose binding domain; a strep tag peptide, such as thatavailable from Biometra in Tampa, Fla.; and an S10 peptide.

The present invention also includes mimetopes of flea allantoinaseproteins of the present invention. As used herein, a mimetope of a fleaallantoinase protein of the present invention refers to any compoundthat is able to mimic the activity of such a flea allantoinase protein,often because the mimetope has a structure that mimics the particularflea allantoinase protein. Mimetopes can be, but are not limited to:peptides that have been modified to decrease their susceptibility todegradation such as all-D retro peptides; anti-idiotypic and/orcatalytic antibodies, or fragments thereof; non-proteinaceousimmunogenic portions of an isolated protein (e.g., carbohydratestructures); and synthetic or natural organic molecules, includingnucleic acids. Such mimetopes can be designed using computer-generatedstructures of proteins of the present invention. Mimetopes can also beobtained by generating random samples of molecules, such asoligonucleotides, peptides or other organic molecules, and screeningsuch samples by affinity chromatography techniques using thecorresponding binding partner.

Another embodiment of the present invention is an isolated nucleic acidmolecule comprising a flea allantoinase nucleic acid molecule, i.e. anucleic acid molecule that can be isolated from a flea cDNA library. Asused herein, flea allantoinase nucleic acid molecules has the samemeaning as flea allantoinase nucleic acid molecule. The identifyingcharacteristics of such nucleic acid molecules are heretofore described.A nucleic acid molecule of the present invention can include an isolatednatural flea allantoinase gene or a homologue thereof, the latter ofwhich is described in more detail below. A nucleic acid molecule of thepresent invention can include one or more regulatory regions,full-length or partial coding regions, or combinations thereof. Theminimal size of a nucleic acid molecule of the present invention is asize sufficient to allow the formation of a stable hybrid (i.e.,hybridization under stringent hybridization conditions) with thecomplementary sequence of another nucleic acid molecule. As such, theminimal size of a flea allantoinase nucleic acid molecule of the presentinvention is from about 12 to about 18 nucleotides in length.

In accordance with the present invention, an isolated nucleic acidmolecule is a nucleic acid molecule that has been removed from itsnatural milieu (i.e., that has been subjected to human manipulation) andcan include DNA, RNA, or derivatives of either DNA or RNA. As such,“isolated” does not reflect the extent to which the nucleic acidmolecule has been purified. Isolated flea allantoinase nucleic acidmolecules of the present invention, or homologues thereof, can beisolated from a natural source or produced using recombinant DNAtechnology (e.g., polymerase chain reaction (PCR) amplification orcloning) or chemical synthesis. Isolated flea allantoinase nucleic acidmolecules, and homologues thereof, can include, for example, naturalallelic variants and nucleic acid molecules modified by nucleotideinsertions, deletions, substitutions, and/or inversions in a manner suchthat the modifications do not substantially interfere with the nucleicacid molecule's ability to encode a flea allantoinase protein of thepresent invention.

A flea allantoinase nucleic acid molecule homologue can be producedusing a number of methods known to those skilled in the art, see, forexample, Sambrook et al., ibid., which is incorporated by referenceherein in its entirety. For example, nucleic acid molecules can bemodified using a variety of techniques including, but not limited to,classic mutagenesis and recombinant DNA techniques such as site-directedmutagenesis, chemical treatment, restriction enzyme cleavage, ligationof nucleic acid fragments, PCR amplification, synthesis ofoligonucleotide mixtures and ligation of mixture groups to “build” amixture of nucleic acid molecules, and combinations thereof. Nucleicacid molecule homologues can be selected by hybridization with fleaallantoinase nucleic acid molecules or by screening the function of aprotein encoded by the nucleic acid molecule (e.g., ability to elicit animmune response against at least one epitope of a flea allantoinaseprotein or to effect flea allantoinase activity).

An isolated flea allantoinase nucleic acid molecule of the presentinvention can include a nucleic acid sequence that encodes at least oneflea allantoinase protein of the present invention respectively,examples of such proteins being disclosed herein. Although the phrase“nucleic acid molecule” primarily refers to the physical nucleic acidmolecule and the phrase “nucleic acid sequence” primarily refers to thesequence of nucleotides on the nucleic acid molecule, the two phrasescan be used interchangeably, especially with respect to a nucleic acidmolecule, or a nucleic acid sequence, being capable of encoding a fleaallantoinase protein.

A preferred nucleic acid molecule of the present invention, whenadministered to an animal, is capable of protecting that animal fromflea infestation. As will be disclosed in more detail below, a nucleicacid molecule of the present invention can be, or encode, an antisenseRNA, a molecule capable of triple helix formation, a ribozyme, or othernucleic acid-based drug compound. In additional embodiments, a nucleicacid molecule of the present invention can encode a protective protein(e.g., a flea allantoinase protein of the present invention), thenucleic acid molecule being delivered to the animal, for example, bydirect injection (i.e, as a genetic vaccine) or in a vehicle such as arecombinant virus vaccine or a recombinant cell vaccine.

In one embodiment of the present invention, a preferred fleaallantoinase nucleic acid molecule includes an isolated nucleic acidmolecule that hybridizes under conditions that preferably allow lessthan or equal to 30% base pair mismatch, preferably under conditionsthat allow less than or equal to 20% base pair mismatch, preferablyunder conditions that allow less than or equal to 10% base pair mismatchpreferably under conditions that allow less than or equal to 5% basepair mismatch or preferably under conditions that allow less than orequal to 2% base pair mismatch with a nucleic acid molecule selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:11.

Another embodiment of the present invention includes a flea allantoinasenucleic acid molecule, wherein said nucleic acid molecule hybridizesunder conditions comprising, (a) hybridizing in solution comprising1×SSC in the absence of nucleic acid helix destabilizing compounds, at atemperature of 37° C. and (b) washing in a solution comprising 1×SSC inthe absence of nucleic acid helix destabilizing compounds, at atemperature of 47° C., to an isolated nucleic acid molecule selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:11.Additional preferred nucleic acid molecules of the present inventioninclude oligonucleotides of an isolated nucleic acid molecule, whereinsaid nucleic acid molecule hybridizes under conditions comprising, (a)hybridizing in solution comprising 1×SSC in the absence of nucleic acidhelix destabilizing compounds, at a temperature of 37° C. and (b)washing in a solution comprising 1×SSC in the absence of nucleic acidhelix destabilizing compounds, at a temperature of 47° C., to anisolated nucleic acid molecule selected from the group consisting of SEQID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:9, and SEQ ID NO:11, wherein said oligonucleotidecomprises at least 30 nucleotides.

Additional preferred flea allantoinase nucleic acid molecules of thepresent invention include nucleic acid molecules comprising a nucleicacid sequence that is preferably at least 70%, preferably at least 80%,preferably at least 90%, preferably at least 92%, preferably at least95%, or preferably at least 98% identical to a nucleic acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, and SEQ IDNO:11. Also preferred are oligonucleotides of any of such nucleic acidmolecules. Percent identity as used herein is determined using theNeedleman-Wunsch algorithm, available in the SeqLab software usingdefault parameters.

One embodiment of the present invention is a nucleic acid moleculecomprising all or part of nucleic acid molecules nCfALN₂₀₃₅, nCfALN₁₄₄₉,nCfALN₁₃₈₃, and nCfALN₁₁₂₃, or allelic variants of these nucleic acidmolecules. Another preferred nucleic acid molecule of the presentinvention includes at least a portion of nucleic acid sequence SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:9, and SEQ ID NO:11, as well as allelic variants of nucleicacid molecules having these nucleic acid sequences and homologues ofnucleic acid molecules having these nucleic acid sequences; preferablysuch a homologue encodes or is complementary to a nucleic acid moleculethat encodes at least one epitope that elicits an immune responseagainst a protein having an amino acid sequence SEQ ID NO:2, SEQ ID NO:7and SEQ ID NO:10. Such nucleic acid molecules can include nucleotides inaddition to those included in the SEQ ID NOs, such as, but not limitedto, a full-length gene, a full-length coding region, a nucleic acidmolecule encoding a fusion protein, or a nucleic acid molecule encodinga multivalent protective compound.

In one embodiment, a flea allantoinase nucleic acid molecule of thepresent invention encodes a protein having an amino acid sequence thatis at least 70%, preferably at least 80%, preferably at least 90%,preferably at least 95%, preferably at least 98%, preferably at least99%, or preferably at least 100% identical to SEQ ID NO:2, SEQ ID NO:7and SEQ ID NO:10. The present invention also includes a fleaallantoinase nucleic acid molecule encoding a protein having at least aportion of SEQ ID NO:2, SEQ ID NO:7 and SEQ ID NO:10, as well as allelicvariants of a nucleic acid molecule encoding a protein having thesesequences, including nucleic acid molecules that have been modified toaccommodate codon usage properties of the cells in which such nucleicacid molecules are to be expressed.

In another embodiment, a preferred flea allantoinase nucleic acidmolecule of the present invention comprises a nucleic acid moleculecomprising at least 20 nucleotides, preferably at least 25 nucleotides,preferably at least 30 nucleotides, preferably at least 40 nucleotides,preferably at least 50 nucleotides, preferably at least 75 nucleotides,preferably at least 100 nucleotides, preferably at least 200nucleotides, preferably at least 400 nucleotides, preferably at least500 nucleotides, preferably at least 750 nucleotides, preferably atleast 1000 nucleotides, preferably at least 1500 nucleotides, preferablyat least 1800 nucleotides, preferably at least 2000 nucleotides, orpreferably at least 2035 nucleotides in length.

In another embodiment, a preferred flea allantoinase nucleic acidmolecule encodes a protein comprising at least 6 amino acids, preferablyat least 10 amino acids, preferably at least 20 amino acids, preferablyat least 30 amino acids, preferably at least 40 amino acids, preferablyat least 50 amino acids, preferably at least 75 amino acids, preferablyat least 100 amino acids, preferably at least 125 amino acids,preferably at least 150 amino acids, preferably at least 175 aminoacids, preferably at least 200 amino acids, preferably at least 250amino acids, preferably at least 300 amino acids, preferably at least350 amino acids, preferably at least 400 amino acids, preferably atleast 450 amino acids, preferably at least 460 amino acids, orpreferably at least 483 amino acids.

In another embodiment, a preferred flea allantoinase nucleic acidmolecule of the present invention comprises an apparently full-lengthflea allantoinase coding region, i.e., the preferred nucleic acidmolecule encodes an apparently full-length flea allantoinase protein,respectively, or a post-translationally modified protein thereof. In oneembodiment, a preferred flea allantoinase nucleic acid molecule of thepresent invention encodes a mature protein.

In another embodiment, a preferred flea allantoinase nucleic acidmolecule of the present invention comprises a nucleic acid moleculecomprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:11, or a fragment thereof.

A fragment of a flea allantoinase nucleic acid molecule of the presentinvention preferably comprises at least 18 nucleotides, preferably atleast 21 nucleotides, preferably at least 25 nucleotides, preferably atleast 30 nucleotides, preferably at least 35 nucleotides, preferably atleast 40 nucleotides, preferably at least 50 nucleotides, preferably atleast 75 nucleotides, preferably at least 100 nucleotides, preferably atleast 200 nucleotides, preferably at least 400 nucleotides, preferablyat least 500 nucleotides, preferably at least 750 nucleotides,preferably at least 1000 nucleotides, preferably at least 1500nucleotides, preferably at least 1800 nucleotides, preferably at least2000 nucleotides, or preferably at least 2035 nucleotides identical insequence to a corresponding contiguous sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:11.

The phrase, a nucleic acid molecule comprising at least “x” contiguous,or consecutive nucleotides identical in sequence to at least “x”contiguous, or consecutive nucleotides of a nucleic acid moleculeselected from the group consisting of SEQ ID NO:“y”, refers to an“x”-nucleotide in length nucleic acid molecule that is identical insequence to an “x”-nucleotide portion of SEQ ID NO:“y”, as well as tonucleic acid molecules that are longer in length than “x”. Theadditional length may be in the form of nucleotides that extend fromeither the 5′ or the 3′ end(s) of the contiguous identical“x”-nucleotide portion. The 5′ and/or 3′ extensions can include one ormore extensions that have no identity to a molecule of the presentinvention, as well as extensions that show similarity or identity tocited nucleic acids sequences or portions thereof.

Knowing the nucleic acid sequences of certain flea allantoinase nucleicacid molecules of the present invention allows one skilled in the artto, for example, (a) make copies of those nucleic acid molecules, (b)obtain nucleic acid molecules including at least a portion of suchnucleic acid molecules (e.g., nucleic acid molecules includingfull-length genes, full-length coding regions, regulatory controlsequences, truncated coding regions), and (c) obtain other fleaallantoinase nucleic acid molecules. Such nucleic acid molecules can beobtained in a variety of ways including screening appropriate expressionlibraries with antibodies of the present invention; traditional cloningtechniques using oligonucleotide probes of the present invention toscreen appropriate libraries; and PCR amplification of appropriatelibraries or DNA using oligonucleotide primers of the present invention.Preferred libraries to screen or from which to amplify nucleic acidmolecules include cDNA libraries as well as genomic DNA libraries.Similarly, preferred DNA sources to screen or from which to amplifynucleic acid molecules include cDNA and genomic DNA. Techniques to cloneand amplify genes are disclosed, for example, in Sambrook et al., ibid.

The present invention also includes nucleic acid molecules that areoligonucleotides capable of hybridizing, under stringent hybridizationconditions, with complementary regions of other, preferably longer,nucleic acid molecules of the present invention such as those comprisingC. felis allantoinase nucleic acid molecules or other flea allantoinasenucleic acid molecules. Oligonucleotides of the present invention can beRNA, DNA, or derivatives of either. The minimum size of sucholigonucleotides is the size required for formation of a stable hybridbetween an oligonucleotide and a complementary sequence on a nucleicacid molecule of the present invention. A preferred oligonucleotide ofthe present invention has a maximum size of preferably 100 to 200nucleotides. The present invention includes oligonucleotides that can beused as, for example, probes to identify nucleic acid molecules, primersto produce nucleic acid molecules, or therapeutic reagents to inhibitflea allantoinase protein production or activity (e.g., as antisense-,triplex formation-, ribozyme- and/or RNA drug-based reagents). Thepresent invention also includes the use of such oligonucleotides toprotect animals from disease using one or more of such technologies.Appropriate oligonucleotide-containing therapeutic compositions can beadministered to an animal using techniques known to those skilled in theart.

One embodiment of the present invention includes a recombinant vector,which includes at least one isolated nucleic acid molecule of thepresent invention, inserted into any vector capable of delivering thenucleic acid molecule into a host cell. Such a vector containsheterologous nucleic acid sequences, that is nucleic acid sequences thatare not naturally found adjacent to nucleic acid molecules of thepresent invention and that preferably are derived from a species otherthan the species from which the nucleic acid molecule(s) are derived.The vector can be either RNA or DNA, either prokaryotic or eukaryotic,and typically is a virus or a plasmid. Recombinant vectors can be usedin the cloning, sequencing, and/or otherwise manipulating of fleaallantoinase nucleic acid molecules of the present invention.

One type of recombinant vector, referred to herein as a recombinantmolecule, comprises a nucleic acid molecule of the present inventionoperatively linked to an expression vector. The phrase operativelylinked refers to insertion of a nucleic acid molecule into an expressionvector in a manner such that the molecule is able to be expressed whentransformed into a host cell. As used herein, an expression vector is aDNA or RNA vector that is capable of transforming a host cell and ofeffecting expression of a specified nucleic acid molecule. Preferably,the expression vector is also capable of replicating within the hostcell. Expression vectors can be either prokaryotic or eukaryotic, andare typically viruses or plasmids. Expression vectors of the presentinvention include any vectors that function (i.e., direct geneexpression) in recombinant cells of the present invention, including inbacterial, fungal, parasite, insect, other animal, and plant cells.Preferred expression vectors of the present invention can direct geneexpression in bacterial, yeast, insect and mammalian cells, and morepreferably in the cell types disclosed herein.

In particular, expression vectors of the present invention containregulatory sequences such as transcription control sequences,translation control sequences, origins of replication, and otherregulatory sequences that are compatible with the recombinant cell andthat control the expression of nucleic acid molecules of the presentinvention. In particular, recombinant molecules of the present inventioninclude transcription control sequences. Transcription control sequencesare sequences that control the initiation, elongation, and terminationof transcription. Particularly important transcription control sequencesare those which control transcription initiation, such as promoter,enhancer, operator and repressor sequences. Suitable transcriptioncontrol sequences include any transcription control sequence that canfunction in at least one of the recombinant cells of the presentinvention. A variety of such transcription control sequences are knownto those skilled in the art. Preferred transcription control sequencesinclude those that function in bacterial, yeast, or insect and mammaliancells, such as, but not limited to, tac, lac, trp, trc, oxy-pro,omp/lpp, rrnB, bacteriophage lambda (such as lambda p_(L) and lambda PRand fusions that include such promoters), bacteriophage T7, T7lac,bacteriophage T3, bacteriophage SP6, bacteriophage SP01,metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirussubgenomic promoter, antibiotic resistance gene, baculovirus, Heliothiszea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, otherpoxvirus, adenovirus, cytomegalovirus (such as immediate earlypromoter), simian virus 40, retrovirus, actin, retroviral long terminalrepeat, Rous sarcoma virus, heat shock, phosphate and nitratetranscription control sequences as well as other sequences capable ofcontrolling gene expression in prokaryotic or eukaryotic cells.Additional suitable transcription control sequences includetissue-specific promoters and enhancers as well as lymphokine-induciblepromoters (e.g., promoters inducible by interferons or interleukins).Transcription control sequences of the present invention can alsoinclude naturally occurring transcription control sequences naturallyassociated with fleas, such as C. felis transcription control sequences.

Suitable and preferred nucleic acid molecules to include in recombinantvectors of the present invention are as disclosed herein. Preferrednucleic acid molecules to include in recombinant vectors, andparticularly in recombinant molecules, include nCfALN₂₀₃₅, nCfALN₁₄₄₉,nCfALN₁₃₈₃, and nCfALN₁₁₂₃.

Recombinant molecules of the present invention may also (a) containsecretory signals (i.e., signal segment nucleic acid sequences) toenable an expressed flea allantoinase protein of the present inventionto be secreted from the cell that produces the protein and/or (b)contain fusion sequences which lead to the expression of nucleic acidmolecules of the present invention as fusion proteins. Examples ofsuitable signal segments include any signal segment capable of directingthe secretion of a protein of the present invention. Preferred signalsegments include, but are not limited to, tissue plasminogen activator(t-PA), interferon, interleukin, growth hormone, histocompatibility andviral envelope glycoprotein signal segments. Suitable fusion segmentsencoded by fusion segment nucleic acids are disclosed herein. Inaddition, a nucleic acid molecule of the present invention can be joinedto a fusion segment that directs the encoded protein to the proteosome,such as a ubiquitin fusion segment. Eukaryotic recombinant molecules mayalso include intervening and/or untranslated sequences surroundingand/or within the nucleic acid sequences of nucleic acid molecules ofthe present invention.

Another embodiment of the present invention includes a recombinant cellcomprising a host cell transformed with one or more recombinantmolecules of the present invention. Transformation of a nucleic acidmolecule into a cell can be accomplished by any method by which anucleic acid molecule can be inserted into the cell. Transformationtechniques include, but are not limited to, transfection,electroporation, microinjection, lipofection, adsorption, and protoplastfusion. A recombinant cell may remain unicellular or may grow into atissue, organ or a multicellular organism. It is to be noted that a cellline refers to any recombinant cell of the present invention that is nota transgenic animal. Transformed nucleic acid molecules of the presentinvention can remain extrachromosomal or can integrate into one or moresites within a chromosome of the transformed (i.e., recombinant) cell insuch a manner that their ability to be expressed is retained. Preferrednucleic acid molecules with which to transform a cell include fleaallantoinase nucleic acid molecules disclosed herein. Preferred nucleicacid molecules with which to transform a cell include nCfALN₂₀₃₅,nCfALN₁₄₄₉, nCfALN₁₃₈₃, and nCfALN_(1123.)

Suitable host cells to transform include any cell that can betransformed with a nucleic acid molecule of the present invention. Hostcells can be either untransformed cells or cells that are alreadytransformed with at least one nucleic acid molecule (e.g., nucleic acidmolecules encoding one or more proteins of the present invention and/orother proteins useful in the production of multivalent vaccines). Hostcells of the present invention either can be endogenously (i.e.,naturally) capable of producing flea allantoinase proteins of thepresent invention or can be capable of producing such proteins afterbeing transformed with at least one nucleic acid molecule of the presentinvention. Host cells of the present invention can be any cell capableof producing at least one protein of the present invention, and includebacterial, fungal (including yeast), parasite (including helminth,protozoa and ectoparasite), other insect, other animal and plant cells.Preferred host cells include bacterial, mycobacterial, yeast, insect andmammalian cells. More preferred host cells include Salmonella,Escherichia, Bacillus, Caulobacter, Listeria, Saccharomyces, Pichia,Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells,MDCK cells (Madin-Darby canine kidney cell line), CRFK cells (Crandellfeline kidney cell line), CV-1 cells (African monkey kidney cell lineused, for example, to culture raccoon poxvirus), COS (e.g., COS-7)cells, and Vero cells. Particularly preferred host cells are Escherichiacoli, including E. coli K-12 derivatives; Salmonella typhi; Salmonellatyphimurium, including attenuated strains such as UK-1_(χ)3987 andSR-11_(χ)4072; Caulobacter; Pichia; Spodoptera frugiperda; Trichoplusiani; BHK cells; MDCK cells; CRFK cells; CV-1 cells; COS cells; Verocells; and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL1246). Additional appropriate mammalian cell hosts include other kidneycell lines, other fibroblast cell lines (e.g., human, murine or chickenembryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovarycells, mouse NIH/3T3 cells, LMTK³¹ cells and/or HeLa cells. In oneembodiment, the proteins may be expressed as heterologous proteins inmyeloma cell lines employing immunoglobulin promoters.

A recombinant cell is preferably produced by transforming a host cellwith one or more recombinant molecules, each comprising one or morenucleic acid molecules of the present invention operatively linked to anexpression vector containing one or more transcription controlsequences, examples of which are disclosed herein. The phraseoperatively linked refers to insertion of a nucleic acid molecule intoan expression vector in a manner such that the molecule is able to beexpressed when transformed into a host cell.

A recombinant cell of the present invention includes any celltransformed with at least one of any nucleic acid molecule of thepresent invention. Suitable and preferred nucleic acid molecules as wellas suitable and preferred recombinant molecules with which to transfercells are disclosed herein.

Recombinant cells of the present invention can also be co-transformedwith one or more recombinant molecules including flea allantoinasenucleic acid molecules encoding one or more proteins of the presentinvention and one or more other nucleic acid molecules encoding otherprotective compounds, as disclosed herein (e.g., to produce multivalentvaccines).

Recombinant DNA technologies can be used to improve expression oftransformed nucleic acid molecules by manipulating, for example, thenumber of copies of the nucleic acid molecules within a host cell, theefficiency with which those nucleic acid molecules are transcribed, theefficiency with which the resultant transcripts are translated, and theefficiency of post-translational modifications. Recombinant techniquesuseful for increasing the expression of nucleic acid molecules of thepresent invention include, but are not limited to, operatively linkingnucleic acid molecules to high-copy number plasmids, integration of thenucleic acid molecules into one or more host cell chromosomes, additionof vector stability sequences to plasmids, substitutions ormodifications of transcription control signals (e.g., promoters,operators, enhancers), substitutions or modifications of translationalcontrol signals (e.g., ribosome binding sites, Shine-Dalgarnosequences), modification of nucleic acid molecules of the presentinvention to correspond to the codon usage of the host cell, deletion ofsequences that destabilize transcripts, and use of control signals thattemporally separate recombinant cell growth from recombinant enzymeproduction during fermentation. The activity of an expressed recombinantprotein of the present invention may be improved by fragmenting,modifying, or derivatizing nucleic acid molecules encoding such aprotein.

Isolated flea allantoinase proteins of the present invention can beproduced in a variety of ways, including production and recovery ofnatural proteins, production and recovery of recombinant proteins, andchemical synthesis of the proteins. In one embodiment, an isolatedprotein of the present invention is produced by culturing a cell capableof expressing the protein under conditions effective to produce theprotein, and recovering the protein. A preferred cell to culture is arecombinant cell of the present invention. Effective culture conditionsinclude, but are not limited to, effective media, bioreactor,temperature, pH and oxygen conditions that permit protein production. Aneffective, medium refers to any medium in which a cell is cultured toproduce a flea allantoinase protein of the present invention. Suchmedium typically comprises an aqueous medium having assimilable carbon,nitrogen and phosphate sources, and appropriate salts, minerals, metalsand other nutrients, such as vitamins. Cells of the present inventioncan be cultured in conventional fermentation bioreactors, shake flasks,test tubes, microtiter dishes, and petri plates. Culturing can becarried out at a temperature, pH and oxygen content appropriate for arecombinant cell. Such culturing conditions are within the expertise ofone of ordinary skill in the art.

Depending on the vector and host system used for production, resultantproteins of the present invention may either remain within therecombinant cell; be secreted into the fermentation medium; be secretedinto a space between two cellular membranes, such as the periplasmicspace in E. coli; or be retained on the outer surface of a cell or viralmembrane.

The phrase “recovering the protein”, as well as similar phrases, refersto collecting the whole fermentation medium containing the protein andneed not imply additional steps of separation or purification. Proteinsof the present invention can be purified using a variety of standardprotein purification techniques, such as, but not limited to, affinitychromatography, ion exchange chromatography, filtration,electrophoresis, hydrophobic interaction chromatography, gel filtrationchromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and differential solubilization.Proteins of the present invention are preferably retrieved in“substantially pure” form. As used herein, “substantially pure” refersto a purity that allows for the effective use of the protein as atherapeutic composition or diagnostic. A therapeutic composition foranimals, for example, should exhibit no substantial toxicity andpreferably should be capable of stimulating the production of antibodiesin a treated animal.

The present invention also includes isolated (i.e., removed from theirnatural milieu) antibodies that selectively bind to a flea allantoinaseprotein of the present invention or a mimetope thereof (e.g., anti-fleaallantoinase antibodies). As used herein, the term “selectively bindsto” a protein refers to the ability of antibodies of the presentinvention to preferentially bind to specified proteins and mimetopesthereof of the present invention. Binding can be measured using avariety of methods standard in the art including enzyme immunoassays(e.g., ELISA), immunoblot assays, etc.; see, for example, Sambrook etal., ibid., and Harlow, et al., 1988, Antibodies, a Laboratory Manual,Cold Spring Harbor Labs Press; Harlow et al., ibid., is incorporated byreference herein in its entirety. An anti-flea allantoinase antibody ofthe present invention preferably selectively binds to a fleaallantoinase protein, respectively, in such a way as to inhibit thefunction of that protein.

Isolated antibodies of the present invention can include antibodies inserum, or antibodies that have been purified to varying degrees.Antibodies of the present invention can be polyclonal or monoclonal, orcan be functional equivalents such as antibody fragments andgenetically-engineered antibodies, including single chain antibodies orchimeric antibodies that can bind to one or more epitopes.

A preferred method to produce antibodies of the present inventionincludes (a) administering to an animal an effective amount of aprotein, peptide or mimetope thereof of the present invention to producethe antibodies and (b) recovering the antibodies. In another method,antibodies of the present invention are produced recombinantly usingtechniques as heretofore disclosed to produce flea allantoinase proteinsof the present invention. Antibodies raised against defined proteins ormimetopes can be advantageous because such antibodies are notsubstantially contaminated with antibodies against other substances thatmight otherwise cause interference in a diagnostic assay or side effectsif used in a therapeutic composition.

Antibodies of the present invention have a variety of potential usesthat are within the scope of the present invention. For example, suchantibodies can be used (a) as therapeutic compounds to passivelyimmunize an animal in order to protect the animal from fleas susceptibleto treatment by such antibodies and/or (b) as tools to screen expressionlibraries and/or to recover desired proteins of the present inventionfrom a mixture of proteins and other contaminants. Furthermore,antibodies of the present invention can be used to target cytotoxicagents to fleas in order to directly kill such fleas. Targeting can beaccomplished by conjugating (i.e., stably joining) such antibodies tothe cytotoxic agents using techniques known to those skilled in the art.Suitable cytotoxic agents are known to those skilled in the art.

One embodiment of the present invention is a therapeutic compositionthat, when administered to an animal susceptible to flea infestation, iscapable of protecting that animal from flea infestation. Therapeuticcompositions of the present invention include at least one of thefollowing protective molecules: an isolated flea allantoinase protein; amimetope of an isolated flea allantoinase protein; an isolated fleaallantoinase nucleic acid molecule; and/or a compound derived from saidisolated flea allantoinase protein that inhibits flea allantoinaseprotein activity. A therapeutic composition of the present invention canfurther comprise a component selected from the group of an excipient, acarrier, and/or an adjuvant; these components are described furtherherein. As used herein, a protective molecule or protective compoundrefers to a compound that, when administered to an animal in aneffective manner, is able to treat, ameliorate, and/or prevent fleainfestation. Preferred fleas to target are heretofore disclosed. Oneexample of a protective molecule is a vaccine, such as, but not limitedto, a naked nucleic acid vaccine, a recombinant virus vaccine, arecombinant cell vaccine, and a recombinant protein vaccine. Anotherexample of a protective molecule is a compound that inhibits fleaallantoinase protein activity, such as an isolated antibody thatselectively binds to a flea allantoinase protein, a substrate analog ofa flea allantoinase protein, anti-sense-, triplex formation-, ribozyme-,and/or RNA drug-based compounds, or other inorganic or organic moleculesthat inhibit flea allantoinase protein activity. Inhibiting fleaallantoinase protein activity can refer to the ability of a compound toreduce the activity of flea allantoinase proteins. Inhibiting fleaallantoinase protein activity can also refer to the ability of acompound to reduce the amount of flea allantoinase protein in a flea.

One embodiment of the present invention is a therapeutic compositioncomprising an excipient and a compound selected from the groupconsisting of: (a) an isolated nucleic acid molecule selected from thegroup consisting of a flea cDNA molecule and a flea mRNA molecule,wherein said nucleic acid molecule is at least 30 nucleotides in lengthand hybridizes with a nucleic acid molecule having a nucleic acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, and SEQID NO:11, under conditions comprising (1) hybridizing in a solutioncomprising 1×SSC in the absence of nucleic acid helix destabilizingcompounds, at a temperature of 37° C. and (2) washing in a solutioncomprising 1×SSC in the absence of helix destabilizing compounds, at atemperature of 47° C.; (b) an isolated protein encoded by a nucleic acidmolecule at least 30 nucleotides in length that hybridizes with anucleic acid molecule having a nucleic acid sequence selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8 and SEQ IDNO:11, under conditions comprising (i) hybridizing in a solutioncomprising 1×SSC in the absence of helix destabilizing compounds, at atemperature of 37° C. and (ii) washing in a solution comprising 1×SSC inthe absence of helix destabilizing compounds, at a temperature of 47°C.; and (c) an isolated antibody that selectively binds to a protein of(b).

Another embodiment of the present invention includes a method to reduceflea infestation in an animal susceptible to flea infestation. Such amethod includes the step of administering to the animal a therapeuticmolecule comprising a protective compound selected from the groupconsisting of (a) an isolated flea allantoinase protein; (b) a mimetopeof an isolated flea allantoinase protein; (c) an isolated fleaallantoinase nucleic acid molecule; and (d) a compound derived from anisolated flea allantoinase protein that inhibits flea allantoinaseprotein activity.

Therapeutic compositions of the present invention can be administered toany animal susceptible to flea infestation, preferably to mammals, andmore preferably to dogs, cats, humans, ferrets, horses, cattle, sheep,and other pets, economic food animals, work animals and/or zoo animals.Preferred animals to protect against flea infestation include dogs,cats, humans, and ferrets, with dogs and cats being particularlypreferred.

As used herein, the term derived, or the term derived from, refers to apeptide, antibody, mimetope, nucleic acid molecule, or other compoundthat was obtained from or obtained using a flea allantoinase protein ornucleic acid molecule of the present invention. Methods to obtainderivatives from a flea allantoinase molecule of the present inventionare known in the art, and as such include, but are not limited tomolecular modeling of flea allantoinase proteins to determine activesites, and predicting from these active sites smaller fragments and/ormimetopes that retain and/or mimic these active sites, therebyinhibiting flea allantoinase protein activity. Other inhibitors of fleaallantoinase activity can also be obtained in a variety of ways,including but not limited to screening of peptide or small chemicalcompound libraries against flea allantoinase proteins of the presentinvention; and screening of polyclonal or monoclonal antibodies to findantibodies that specifically bind flea allantoinase proteins of thepresent invention.

A flea allantoinase protein inhibitor of the present invention (i.e. aninhibitor of a flea allantoinase protein) is identified by its abilityto mimic, bind to, modify, or otherwise interact with, a fleaallantoinase protein, thereby inhibiting the activity of a natural fleaallantoinase protein. Suitable inhibitors of flea allantoinase proteinactivity are compounds that inhibit flea allantoinase protein activityin at least one of a variety of ways: (a) by binding to or otherwiseinteracting with or otherwise modifying flea allantoinase protein sites;(b) by binding to the flea allantoinase protein and thus reducing theavailability of the flea allantoinase protein in solution; (c) bymimicking a flea allantoinase protein; and (d) by interacting with otherregions of the flea allantoinase protein to inhibit flea allantoinaseprotein activity, for example, by allosteric interaction.

Flea allantoinase protein inhibitors can be used directly as compoundsin compositions of the present invention to treat animals as long assuch compounds are not harmful to host animals being treated. Preferredflea allantoinase protein inhibitors of the present invention include,but are not limited to, flea allantoinase protein substrate analogs, andother molecules that bind to a flea allantoinase protein (e.g., to anallosteric site) in such a manner that the activity of the fleaallantoinase protein is inhibited. A flea allantoinase protein substrateanalog refers to a compound that interacts with (e.g., binds to,associates with, modifies) the active site of a flea allantoinaseprotein. A preferred flea allantoinase protein substrate analog inhibitsflea allantoinase protein activity. Flea allantoinase protein substrateanalogs can be of any inorganic or organic composition. Fleaallantoinase protein substrate analogs can be, but need not be,structurally similar to a flea allantoinase protein natural substrate aslong as they can interact with the active site of that flea allantoinaseprotein. Flea allantoinase protein substrate analogs can be designedusing computer-generated structures of flea allantoinase proteins of thepresent invention or computer structures of flea allantoinase protein'snatural substrates. Preferred sites to model include one or more of theactive sites of flea allantoinase proteins. Substrate analogs can alsobe obtained by generating random samples of molecules, such asoligonucleotides, peptides, peptidomimetic compounds, or other inorganicor organic molecules, and screening such samples for their ability tointerfere with interaction between flea allantoinase proteins and theirsubstrates, e.g. by affinity chromatography techniques. A preferred fleaallantoinase protein substrate analog is a flea allantoinase proteinmimetic compound, i.e., a compound that is structurally and/orfunctionally similar to a natural substrate of a flea allantoinaseprotein of the present invention, particularly to the region of thesubstrate that interacts with the flea allantoinase protein active site,but that inhibits flea allantoinase protein activity upon interactingwith the flea allantoinase protein active site.

The present invention also includes a therapeutic composition comprisingat least one protective molecule of the present invention in combinationwith at least one additional compound protective against one or moreinfectious agents.

In one embodiment, a therapeutic composition of the present inventioncan be used to protect an animal from flea infestation by administeringsuch composition to a flea in order to prevent infestation. Suchadministration to the flea and/or animal could be oral, or byapplication to the animal's body surface (e.g. topical spot-on, orspraying onto the animal), or by application to the environment (e.g.,spraying). Examples of such compositions include, but are not limitedto, transgenic vectors capable of producing at least one therapeuticcomposition of the present invention. In another embodiment a flea caningest therapeutic compositions, or products thereof, present on thesurface of or in the blood of a host animal that has been administered atherapeutic composition of the present invention.

In accordance with the present invention, a host animal (i.e., an animalthat is or is capable of being infested with fleas) is treated byadministering to the animal a therapeutic composition of the presentinvention in such a manner that the composition itself (e.g., a fleaallantoinase protein, a flea allaantoinase nucleic acid molecule, a fleaallantoinase protein inhibitor, a allantoinase protein synthesissuppressor (i.e., a compound that decreases the production or half-lifeof a allantoinase protein in fleas), a flea allantoinase proteinmimetope, or a anti-flea allantoinase antibody) or a product generatedby the animal in response to administration of the composition (e.g.,antibodies produced in response to administration of a flea allantoinaseprotein or nucleic acid molecule, or conversion of an inactive inhibitor“prodrug” to an active flea allantoinase protein inhibitor) ultimatelyenters the flea. A host animal is preferably treated in such a way thatthe compound or product thereof is present on the body surface of theanimal or enters the blood stream of the animal. Fleas are then exposedto the composition or product when they feed from the animal. Forexample, flea allantoinase protein inhibitors administered to an animalare administered in such a way that the inhibitors enter the bloodstream of the animal, where they can be taken up by feeding fleas.

The present invention also includes the ability to reduce larval fleainfestation in that when fleas feed from a host animal that has beenadministered a therapeutic composition of the present invention, atleast a portion of compounds of the present invention, or productsthereof, in the blood taken up by the fleas are excreted by the fleas infeces, which is subsequently ingested by flea larvae. In particular, itis of note that flea larvae obtain most, if not all, of their nutritionfrom flea feces.

In accordance with the present invention, reducing flea allantoinaseprotein activity in a flea can lead to a number of outcomes that reduceflea burden on treated animals and their surrounding environments. Suchoutcomes include, but are not limited to, (a) reducing the viability offleas that feed from the treated animal, (b) reducing the fecundity offemale fleas that feed from the treated animal, (c) reducing thereproductive capacity of male fleas that feed from the treated animal,(d) reducing the viability of eggs laid by female fleas that feed fromthe treated animal, (e) altering the blood feeding behavior of fleasthat feed from the treated animal (e.g., fleas take up less volume perfeeding or feed less frequently), (f) reducing the viability of flealarvae, for example due to the feeding of larvae from feces of fleasthat feed from the treated animal, (g) altering the development of flealarvae (e.g., by decreasing feeding behavior, inhibiting growth,inhibiting (e.g., slowing or blocking) molting, and/or otherwiseinhibiting maturation to adults), and/or (h) altering or decreasing theability of fleas or flea larvae to digest a blood meal.

In order to protect an animal from flea infestation, a therapeuticcomposition of the present invention is administered to the animal in aneffective manner such that the composition is capable of protecting thatanimal from flea infestation. Therapeutic compositions of the presentinvention can be administered to animals prior to infestation in orderto prevent infestation (i.e., as a preventative vaccine) and/or can beadministered to animals after infestation. For example, proteins,mimetopes thereof, and antibodies thereof can be used asimmunotherapeutic agents.

Therapeutic compositions of the present invention can be formulated inan excipient that the animal to be treated can tolerate. Examples ofsuch excipients include water, saline, Ringer's solution, dextrosesolution, Hank's solution, and other aqueous physiologically balancedsalt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil,ethyl oleate, or triglycerides may also be used. Other usefulformulations include suspensions containing viscosity enhancing agents,such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipientscan also contain minor amounts of additives, such as substances thatenhance isotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and Tris buffer, while examples ofpreservatives include thimerosal, or o-cresol, formalin and benzylalcohol. Standard formulations can either be liquid injectables orsolids which can be taken up in a suitable liquid as a suspension orsolution for injection. Thus, in a non-liquid formulation, the excipientcan comprise dextrose, serum albumin, preservatives, etc., to whichsterile water or saline can be added prior to administration.

In one embodiment of the present invention, a therapeutic compositioncan include an adjuvant. Adjuvants are agents that are capable ofenhancing the immune response of an animal to a specific antigen.Suitable adjuvants include, but are not limited to, cytokines,chemokines, and compounds that induce the production of cytokines andchemokines (e.g., granulocyte macrophage colony stimulating factor(GM-CSF), Flt-3 ligand, granulocyte colony stimulating factor (G-CSF),macrophage colony stimulating factor (M-CSF), colony stimulating factor(CSF), erythropoietin (EPO), interleukin 2 (L-2), interleukin-3 (IL-3),interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6),interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10 (IL-10),interleukin 12 (IL-12), interferon gamma, interferon gamma inducingfactor I (IGIF), transforming growth factor beta, RANTES (regulated uponactivation, normal T cell expressed and presumably secreted), macrophageinflammatory proteins (e.g., MIP-1 alpha and MIP-1 beta), and Leishmaniaelongation initiating factor (LEIF)); bacterial components (e.g.,endotoxins, in particular superantigens, exotoxins and cell wallcomponents); aluminum-based salts; calcium-based salts; silica;polynucleotides; toxoids; serum proteins, viral coat proteins; blockcopolymer adjuvants (e.g., Hunter's Titermax™ adjuvant (Vaxcel™, Inc.Norcross, Ga.), Ribi adjuvants (Ribi ImmunoChem Research, Inc.,Hamilton, Mont.); and saponins and their derivatives (e.g., Quil A(Superfos Biosector A/S, Denmark). Protein adjuvants of the presentinvention can be delivered in the form of the protein themselves or ofnucleic acid molecules encoding such proteins using the methodsdescribed herein.

In one embodiment of the present invention, a therapeutic compositioncan include a carrier. Carriers include compounds that increase thehalf-life of a therapeutic composition in the treated animal. Suitablecarriers include, but are not limited to, polymeric controlled releasevehicles, biodegradable implants, liposomes, bacteria, viruses, othercells, oils, esters, and glycols.

One embodiment of the present invention is a controlled releaseformulation that is capable of slowly releasing a composition of thepresent invention into an animal. As used herein, a controlled releaseformulation comprises a composition of the present invention in acontrolled release vehicle. Suitable controlled release vehiclesinclude, but are not limited to, biocompatible polymers, other polymericmatrices, capsules, microcapsules, microparticles, bolus preparations,osmotic pumps, diffusion devices, liposomes, lipospheres, andtransdermal delivery systems. Other controlled release formulations ofthe present invention include liquids that, upon administration to ananimal, form a solid or a gel in situ. Preferred controlled releaseformulations are biodegradable (i.e., bioerodible).

A preferred controlled release formulation of the present invention iscapable of releasing a composition of the present invention into theblood of the treated animal at a constant rate sufficient to attaintherapeutic dose levels of the composition. The therapeutic compositionis preferably released over a period of time ranging from 1 to 12months. A controlled release formulation of the present invention iscapable of effecting a treatment preferably for at least 1 month,preferably for at least 3 months, preferably for at least 6 months,preferably for at least 9 months, and preferably for at least 12 months.

Acceptable protocols to administer therapeutic compositions in aneffective manner include individual dose size, number of doses,frequency of dose administration, and mode of administration.Determination of such protocols can be accomplished by those skilled inthe art. A suitable single dose is a dose that is capable of treating ananimal when administered one or more times over a suitable time period.For example, a preferred single dose of an inhibitor is from about 1microgram (μg) to about 10 milligrams (mg) of the therapeuticcomposition per kilogram body weight of the animal. Booster vaccinationscan be administered from about 2 weeks to several years after theoriginal administration. Booster administrations preferably areadministered when the immune response of the animal becomes insufficientto protect the animal from disease. A preferred administration scheduleis one in which from about 10 μg to about 1 mg of the therapeuticcomposition per kg body weight of the animal is administered from aboutone to about two times over a time period of from about 2 weeks to about12 months. Modes of administration can include, but are not limited to,subcutaneous, intradermal, intravenous, intranasal, oral, transdermal,intraocular, intranasal, conjunctival, and intramuscular routes. Methodsof administration for other therapeutic compounds can be determined byone skilled in the art, and may include administration of a therapeuticcomposition one or more times, on a daily, weekly, monthly or yearlyregimen; routes of administration can be determined by one skilled inthe art, and may include any route. A preferred route of administrationof an inhibitory compound when administering to fleas is a topical, or“spot-on” formulation administered to the body surface of the animal, sothat a flea would encounter the inhibitory compound when attached to theanimal; another preferred route of administration of an inhibitorycompound is an oral formulation that, when fed to an animal, would enterthe bloodstream of the animal, which would then be transferred to a fleawhile feeding from the animal.

A recombinant protein vaccine of the present invention comprises arecombinantly-produced flea allantoinase protein of the presentinvention that is administered to an animal according to a protocol thatresults in the animal producing a sufficient immune response to protectitself from a flea infestation. Such protocols can be determined bythose skilled in the art.

According to one embodiment, a nucleic acid molecule of the presentinvention can be administered to an animal in a fashion to enableexpression of that nucleic acid molecule into a protective protein orprotective RNA (e.g., antisense RNA, ribozyme, triple helix forms or RNAdrug) in the animal. Nucleic acid molecules can be delivered to ananimal in a variety of methods including, but not limited to, (a)administering a naked (i.e., not packaged in a viral coat or cellularmembrane) nucleic acid as a genetic vaccine (e.g., as naked DNA or RNAmolecules, such as is taught, for example in Wolff et al., 1990, Science247, 1465-1468) or (b) administering a nucleic acid molecule packaged asa recombinant virus vaccine or as a recombinant cell vaccine (i.e., thenucleic acid molecule is delivered by a viral or cellular vehicle).

A genetic (i.e., naked nucleic acid) vaccine of the present inventionincludes a nucleic acid molecule of the present invention and preferablyincludes a recombinant molecule of the present invention that preferablyis replication, or otherwise amplification, competent. A genetic vaccineof the present invention can comprise one or more nucleic acid moleculesof the present invention in the form of, for example, a dicistronicrecombinant molecule. Preferred genetic vaccines include at least aportion of a viral genome, i.e., a viral vector. Preferred viral vectorsinclude those based on alphaviruses, poxviruses, adenoviruses,herpesviruses, picornaviruses, and retroviruses, with those based onalphaviruses, such as sindbis or Semliki forest virus, species-specificherpesviruses and poxviruses being particularly preferred. Any suitabletranscription control sequence can be used, including those disclosed assuitable for protein production. Particularly preferred transcriptioncontrol sequences include cytomegalovirus immediate early (preferably inconjunction with Intron-A), Rous sarcoma virus long terminal repeat, andtissue-specific transcription control sequences, as well astranscription control sequences endogenous to viral vectors if viralvectors are used. The incorporation of a “strong” polyadenylation signalis also preferred.

Genetic vaccines of the present invention can be administered in avariety of ways, with intramuscular, subcutaneous, intradermal,transdermal, conjunctival, intraocular, intranasal and oral routes ofadministration being preferred. A preferred single dose of a geneticvaccine ranges from about 1 nanogram (ng) to about 600 μg, depending onthe route of administration and/or method of delivery, as can bedetermined by those skilled in the art. Suitable delivery methodsinclude, for example, by injection, as drops, aerosolized and/ortopically. Genetic vaccines of the present invention can be contained inan aqueous excipient (e.g., phosphate buffered saline) alone or in acarrier (e.g., lipid-based vehicles).

A recombinant virus vaccine of the present invention includes arecombinant molecule of the present invention that is packaged in aviral coat and that can be expressed in an animal after administration.Preferably, the recombinant molecule is packaging- orreplication-deficient and/or encodes an attenuated virus. A number ofrecombinant viruses can be used, including, but not limited to, thosebased on alphaviruses, poxviruses, adenoviruses, herpesviruses,picornaviruses, and retroviruses. Preferred recombinant virus vaccinesare those based on alphaviruses (such as Sindbis virus), raccoonpoxviruses, species-specific herpesviruses and species-specificpoxviruses. An example of methods to produce and use alphavirusrecombinant virus vaccines are disclosed in U.S. Pat. No. 5,766,602 toXiong and Grieve, which is incorporated by reference herein in itsentirety.

When administered to an animal, a recombinant virus vaccine of thepresent invention infects cells within the immunized animal and directsthe production of a protective protein or RNA nucleic acid molecule thatis capable of protecting the animal from flea infestation as disclosedherein. For example, a recombinant virus vaccine comprising a fleaallantoinase nucleic acid molecule of the present invention isadministered according to a protocol that results in the animalproducing a sufficient immune response to protect itself from fleainfestation. A preferred single dose of a recombinant virus vaccine ofthe present invention is from about 1×10⁴ to about 1×10⁸ virus plaqueforming units (pfu) per kilogram body weight of the animal.Administration protocols are similar to those described herein forprotein-based vaccines, with subcutaneous, intramuscular, intranasal,intraocular, conjunctival, and oral administration routes beingpreferred.

A recombinant cell vaccine of the present invention includes recombinantcells of the present invention that express at least one protein of thepresent invention. Preferred recombinant cells for this embodimentinclude Salmonella, E. coli, Listeria, Mycobacterium, S. frugiperda,yeast, (including Saccharomyces cerevisiae and Pichia pastoris), BHK,CV-1, myoblast G8, COS (e.g., COS-7), Vero, MDCK and CRFK recombinantcells. Recombinant cell vaccines of the present invention can beadministered in a variety of ways but have the advantage that they canbe administered orally, preferably at doses ranging from about 10⁸ toabout 10¹² cells per kilogram body weight. Administration protocols aresimilar to those described herein for protein-based vaccines.Recombinant cell vaccines can comprise whole cells, cells stripped ofcell walls or cell lysates.

The efficacy of a therapeutic composition of the present invention toprotect an animal from flea infestation can be tested in a variety ofways including, but not limited to, detection of protective antibodies(using, for example, proteins or mimetopes of the present invention),detection of cellular immunity within the treated animal, or challengeof the treated animal with the fleas to determine whether the treatedanimal is resistant to infestation. Challenge studies can include directadministration of fleas to the treated animal. In one embodiment,therapeutic compositions can be tested in animal models such as mice.Such techniques are known to those skilled in the art.

As discussed herein, one therapeutic composition of the presentinvention includes an inhibitor of flea allantoinase protein activity,i.e., a compound capable of substantially interfering with the functionof a flea allantoinase protein. An inhibitor of flea allantoinaseprotein activity, or function, can be identified using flea allantoinaseproteins of the present invention. A preferred inhibitor of fleaallantoinase protein function is a compound capable of substantiallyinterfering with the function of a flea allantoinase protein and whichdoes not substantially interfere with the function of host animalallantoinase proteins. As used herein, a compound that does notsubstantially inhibit or interfere with host animal allantoinaseproteins is one that, when administered to a host animal, the hostanimal shows no significant adverse effects attributable to theinhibition of allantoinase and which, when administered to an animal inan effective manner, is capable of protecting that animal from fleainfestation.

One embodiment of the present invention is a method to identify acompound capable of inhibiting flea allantoinase protein activity. Sucha method includes the steps of (a) contacting (e.g., combining, mixing)an isolated flea allantoinase protein of the present invention, such asa flea extract having allantoinase activity, with a putative inhibitorycompound under conditions in which, in the absence of the compound, theprotein has flea allantoinase protein activity, and (b) determining ifthe putative inhibitory compound inhibits the activity. Fleaallantoinase protein activity can be determined in a variety of waysknown in the art, including but not limited to determining the abilityof flea allantoinase protein to bind to or otherwise interact with asubstrate. Such conditions under which a flea allantoinase protein hasflea allantoinase protein activity include conditions in which a fleaallantoinase protein has a correct three-dimensionally folded structureunder physiologic conditions, i.e. physiologic pH, physiologic ionicconcentrations, and physiologic temperatures.

Putative inhibitory compounds to screen include antibodies (includingfragments and mimetopes thereof), putative substrate analogs, and other,preferably small, organic or inorganic molecules. Methods to determineflea allantoinase protein activity are known to those skilled in theart.

A preferred method to identify a compound capable of inhibiting fleaallantoinase protein activity includes contacting an isolated fleaallantoinase protein of the present, invention with a putativeinhibitory compound under conditions in which, in the absence of thecompound, the protein has flea allantoinase protein activity; anddetermining if the putative inhibitory compound inhibits the activity.

Another embodiment of the present invention is an assay kit to identifyan inhibitor of a flea allantoinase protein of the present invention.This kit comprises an isolated flea allantoinase protein of the presentinvention, and a means for determining inhibition of an activity of fleaallantoinase protein, where the means enables detection of inhibition.Detection of inhibition of flea allantoinase protein identifies aputative inhibitor to be an inhibitor of a flea allantoinase protein.Means for determining inhibition of a flea allantoinase protein include,for example, an assay system that detects binding of a putativeinhibitor to a flea allantoinase molecule, and an assay system thatdetects interference by a putative inhibitor of the ability of fleaallantoinase protein to hydrolyze a substrate. Means and methods aredescribed herein and are known to those skilled in the art.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the present invention. Thefollowing examples include a number of recombinant DNA and proteinchemistry techniques known to those skilled in the art; see, forexample, Sambrook et al., ibid.

EXAMPLE 1

This Example describes the isolation of RNA from the hindgut andMalpighian tubules (HMT) of Ctenocephalides felis and the use ofisolated RNA to construct subtracted and unsubtracted cDNA libraries.

Approximately 10,000 hindguts and Malpighian tubules were dissected fromequal numbers of cat blood fed and unfed adult C. felis with a male tofemale ratio of 1 to 4, and total RNA was extracted using a guanidineisothiocyanate lysis buffer and the standard procedure described bySambrook et al. Poly-A enriched mRNA was purified from total RNA aboveusing a mRNA Purification Kit, available from Pharmacia Biotech,Piscataway, N.J., following the manufacturer's protocol. The sameprocedures were used to extract total RNA and isolate poly-A enrichedmRNA from the dissected C. felis bodies following removal of HMT,referred to hereinafter as “non-HMT mRNA”.

Poly-A enriched mRNA was used to construct a cDNA library usingsubtractive hybridization and suppression PCR as follows. Subtractivehybridization and suppression PCR was conducted using a PCR-Select™ cDNASubtraction Kit, available from Clontech Laboratories, Inc., Palo Alto,Calif. according to the manufacturer's instructions. Briefly, this kituses subtractive hybridization and suppression PCR to specificallyamplify cDNA sequences that are present in the tester cDNA and absent inthe driver cDNA, thus enriching for tester-specific sequences. Theefficiency of the subtraction process can be assessed bysemi-quantitative PCR and by comparing the ethidium bromide stainingpatterns of the subtracted and unsubtracted samples on agarose gels asdescribed in section V.D. of the manufacturer's protocol. For thesemi-quantitative PCR, three genes with mRNAs known to be expressedoutside of the HMT tissue were used to test for specific subtraction.These genes encoded putative actin, N-aminopeptidase, and serineprotease proteins.

Subtractive hybridization and suppression PCR was conducted under thefollowing conditions. Two micrograms (μg) of HMT mRNA was used as thetemplate for synthesis of the tester material and 2 μg of non-HMT mRNAwas used as template for synthesis of the driver material in thisreaction. The number of cycles used in the selective amplification stepswas optimized using the manufacturer's protocols. Optimization resultedin the use of 24 rather than the standard 27 cycles of primary PCR incombination with 15 cycles of secondary PCR rather than the standard 12cycles.

The products from the suppressive PCR reaction were ligated into thepCR®2.1 vector, available from Invitrogen, Carlsbad, Calif., using anOriginal TA Cloning® Kit, available from Invitrogen. The ligationreaction was then used to transform INVαF′ One Shot™ competent cells,available from Invitrogen, which were plated on Luria broth (LB) agarwith 50 micrograms per milliliter (μg/ml) ampicillin, available fromSigma-Aldrich Co., St. Louis, Mo., and 50 μg/ml5-bromo-4-chloro-3-indoyl β-D-galactopyranoside (X-Gal), available fromFisher Biotech, Fair Lawn, N.J. Transformed colonies were amplified andthe DNA isolated using the standard alkaline lysis procedure describedby Sambrook et al., ibid.

Automated cycle sequencing of DNA samples was performed using an ABIPRISM™ Model 377, available from Perkins Elmer, with XL upgrade DNASequencer, available from PE Applied Biosystems, Foster City, Calif.,after reactions were carried out using the PRISM™ Dye Terminator CycleSequencing Ready Reaction Kit or the PRISM™ dRhodamine Terminator CycleSequencing Ready Reaction Kit or the PRISM™ BigDye™ Terminator Cyclesequencing Ready Reaction Kit, available from PE Applied Biosystems,following the manufacturer's protocol, hereinafter “standard sequencingmethods”. Sequence analysis was performed using SeqLab, using defaultparameters. Each sequence read was trimmed of vector sequence at eitherend and submitted for a search through the National Center forBiotechnology Information (NCBI), National Library of Medicine, NationalInstitute of Health, Baltimore, Md., using the BLAST network. Thisdatabase includes SwissProt+PIR+SPupdate+GenPept+GPUpdate+PDB databases.The search was conducted using the xBLAST function, which compares thetranslated sequences in all 6 reading frames to the protein sequencescontained in the database.

An unsubtracted HMT cDNA library was constructed as follows.Approximately 10,000 HMT tissues were dissected from equal numbers ofunfed and cat blood-fed adult C. felis with a male to female ratio of1:4. Total RNA was extracted using a guanidine isothiocyanate lysisbuffer and procedures described in Sambrook et al., followed byisolation using a mRNA purification kit, available from Pharmacia,according to the manufacturer's protocols. The library was constructedwith 5 μg of isolated mRNA using a ZAP-cDNA® cDNA synthesis kit, andpackaged using a ZAP-cDNA® Gigapack® gold cloning kit, both availablefrom Stratagene, La Jolla, Calif. The resultant HMT library wasamplified to a titer of about 5×10⁹ plaque forming units per milliliter(pfu/ml). Single clone excisions were performed using the Ex-Assist™helper phage, available from Stratagene, and used to create doublestranded plasmid template for sequencing using the manufacturer'sprotocols with the following exceptions. Following incubation of theSOLR cells with the cleared phage lysate, the mixture was used toinoculate LB broth, and the mix was incubated overnight and thensubjected to mini-prep plasmid preparation and sequencing as describedfor the subtracted HMT library above.

EXAMPLE 2

This example describes the cloning and sequencing of a C. felisallantoinase nucleic acid molecule of the present invention andexpression and purification of recombinant protein therefrom. Thisexample also describes the expression of allantoinase mRNA in a varietyof flea tissues, activity assays of native proteins, the production ofaffinity purified antibody to a recombinant flea allantoinase protein,and protein localization by immunohistochemistry.

A. Isolation of Nucleic Acid Sequences

A TA clone from the HMT EST library described in Example 1 was sequencedusing standard sequencing methods and shown to encode a partialpolypeptide having significant homology to allantoinase proteins. Thisclone was digested with EcoRI to excise an insert 682 nucleotides inlength, referred to as flea nucleic acid molecule nCfALN₆₈₂. The insertwas isolated by gel purification using a Gel Purification kit, availablefrom Qiagen, Chatsworth, Calif. Approximately 50 nanograms (ng) ofpurified nCfALN₆₈₂ was used to construct a ³²P α-dATP labeled DNA probeusing a Megaprime DNA labeling kit, available from Amersham, ArlingtonHeights, Ill., using the manufacturer's protocols.

The ³²P α-dATP labeled probe was used in a plaque lift hybridizationprocedure to isolate a clone from the HMT lambda-ZAP unsubtracted cDNAlibrary described in Example 1 under the following hybridizationconditions. Filters were hybridized with about 1×10⁶ counts per minute(cpm) per ml of the probe in 5×SSPE, (see Sambrook et al., ibid.), 1.2%sodium dodecyl sulfate (SDS), 0.1 mg/ml salmon sperm DNA and5×Denhardt's reagent, (see Sambrook et al., ibid.), at 55° C. for about14 hours. The filters were washed as follows: (a) 10 minutes with 5×SSPEand 1% SDS, (b) 10 minutes with 2×SSPE and 1% SDS, (c) 10 minutes with1×SSPE and 0.5% SDS, and (d) 10 minutes with 0.5×SSPE and 1% SDS. Allwashes were conducted at 55° C. Plaques that hybridized strongly to theprobe were isolated and subjected to in vivo excision. In vivo excisionwas performed using the Stratagene Ex-Assist™ helper phage system andprotocols, to convert a positive plaque to pBluescript™ plasmid DNA.Sequencing was conducted using standard sequencing methods followingpreparation of DNA with a Qiagen Qiaprep™ spin mini prep kit using themanufacturer's instructions and restriction enzyme digestion with about1 μl of 20 U/μl each of EcoRI and XhoI, available from New EnglandBiolabs, Beverly, Mass. A clone was isolated from a primary plaque,containing a nucleic acid molecule of about 2035 base pairs, referred toherein as nCfALN₂₀₃₅, the coding strand of which is a nucleotidesequence denoted herein as SEQ ID NO:1. The complement of SEQ ID NO:1 isrepresented herein as SEQ ID NO:3.

The clone containing SEQ ID NO:1 was originally sequenced and namednCfALN₂₀₅₇, see PCT Publication WO 00/61621. Upon re-sequencing ofnCfALN₂₀₅₇ it was determined that a sequencing error occurred, resultingin the insertion of a guanine at position 1292. SEQ ID NO:1 alsoreflects the removal of a poly-A tail in the 3′ untranslated region ofthe molecule, but otherwise nCfALN₂₀₅₇ and nCfALN₂₀₃₅ are identical.

Translation of SEQ ID NO:1 suggests that nucleic acid moleculenCfALN₂₀₃₅ encodes a full-length allantoinase protein of 483 aminoacids, referred to herein as PCfALN₄₈₃, the amino acid sequence of whichis represented by SEQ ID NO:2, assuming the initiation codon spans fromnucleotide 152 through nucleotide 154 of SEQ ID NO:1 and the terminationcodon spans from nucleotide 1601 through nucleotide 1603 of SEQ ID NO:1.The coding region encoding PCfALN₄₈₃, is represented by nucleic acidmolecule nCfALN₁₄₄₉, having a coding strand with the nucleic acidsequence represented by SEQ ID NO:4 and a complementary strand withnucleic acid sequence represented by SEQ ID NO:5. The amino acidsequence of PCfALN₄₈₃ also represented as SEQ ID NO:2, predicts thatPCfALN₄₈₃ has an estimated molecular weight of about 53 kilodaltons(kDa) and an estimated isoelectric point (pI) of about 6.34.

Comparison of amino acid sequence SEQ ID NO:2 with amino acid sequencesreported in GenBank indicates that SEQ ID NO:2 showed the most homology,i.e., about 45.7% identity, with a Streptomyces coelicolor “probableallantoinase” protein GenBank Accession No. Q9RKU5, and a second highesthomology, i.e. about 45.1% identity to a Rana catesbeiana (bullfrog)allantoinase protein, GenBank Accession No. 458126. Comparison of SEQ IDNO:4 with nucleic acid sequences reported in GenBank indicates that SEQID NO:4 showed the most homology, i.e., about 51% identity, with a Ranacatesbeiana nucleic acid molecule, GenBank Accession number U03471.Percent identity calculations were performed using GCG version 9.0 usingdefault parameters.

B. Protein Expression

The region of nCfALN₂₀₃₅ encoding the predicted mature protein, referredto herein as nCfALN₁₃₈₃, the coding strand of which is a nucleotidesequence designated SEQ ID NO:6 and a complementary strand designatedSEQ ID NO:8, was PCR amplified using the pBluescript™ clone describedabove as the template. Sense primer ALLA-FE, having nucleotide sequence5′ CAT GCC ATG GCG TGC ACC AAC AAC GCG CCT CC 3′, having a NcoI siteindicated in bold, designated herein as SEQ ID NO:12, and anti-senseprimer ALLA-RE, having nucleotide sequence 5′ GCG GTA CCT CAT TCA ATAAGT AAA TTT CCT TTT GG 3′, having a KpnI site indicated in bold,designated herein as SEQ ID NO:13 were used in the PCR reaction. PCRreactions were performed using the following amplification cycles: (a)one cycle at 95° C. for thirty seconds; (b) thirty cycles at 95° C. fortwenty seconds, 50° C. for twenty seconds, and 72° C. for two minutes;and (c) one cycle at 72° C. for five minutes, in reactions containing2.5 mM MgCl₂, 0.2 mM dNTPs, 1 μM of each primer, 0.5 μl of 5U/μl Taqpolymerase, 1 μl of 1 μg/μl template, and 3 μl of 10×Taq buffer. The PCRproduct was digested with NcoI and KpnI and ligated into the vectorpTrcHisB, available from Invitrogen, that had been digested with NcoIand KpnI and treated with alkaline phosphatase. The resultingrecombinant molecule, referred to herein as pTrc-nCfALN₁₃₈₃, wastransformed into E. coli strain BL21, available from Novagen Inc.,Madison, Wis., to form recombinant cell E. coli:pTrc-nCfALN₁₃₈₃.

The recombinant cell was grown under standard conditions and thenincubated in the presence of 0.5 μM isopropylthio-β-galactoside (IPTG)to induce expression of recombinant protein, predicted to beapproximately 50.6 kDa, referred to herein as PCfALN₄₆₁ with an aminoacid sequence designated SEQ ID NO:7. Expression was confirmed usingCoomassie-blue-stained Tris-glycine gel and by Western blot using anaffinity-purified rabbit antibody, the production of which is describedin Section D below, which showed expression of an about 52-kDa protein.

A non-full length protein encoded by nucleotides 187-1308 of nCfALN₂₀₃₅was PCR amplified using the pBluescript™ clone described above as thetemplate. PCR reactions were performed as described above using senseprimer ALN-FE, having nucleotide sequence 5′ GCG GAT CCT ATG CTG AAT TGCAAG AAC CTT G 3′, having a BamHI site indicated in bold, designatedherein as SEQ ID NO:14, and anti-sense primer ALN-RE, having nucleotidesequence 5′ CAG GTA CCC TCT TTT AGA AGC ACC GGT CCC 3′, having a KpnIsite indicated in bold, designated herein as SEQ ID NO:15. The PCRproduct, referred to as nCfALN₁₁₂₃, with a forward strand designated SEQID NO:9 and a reverse strand designated SEQ ID NO:11, was digested withBamHI and KpnI and ligated into the vector pTrcHisB, available fromInvitrogen, that had been digested with BamHI and KpnI and treated withalkaline phosphatase. The resulting recombinant molecule, referred toherein as pTrc-nCfALN₁₁₂₃, was transformed into E. coli strain BL21,available from Novagen Inc., Madison, Wis., to form recombinant cell E.coli:pTrc-nCfALN₁₁₂₃.

The recombinant cell was grown under standard conditions and thenincubated in the presence of 0.5 μM isopropylthio-β-galactoside (IPTG)to induce expression of recombinant protein, predicted to beapproximately 42.2 kDa, referred to herein as PCfALN₃₇₄ with an aminoacid sequence designated SEQ ID NO:10. Expression was confirmed usingCoomassie-blue-stained Tris-glycine gel and by Western blot using a T7tag antibody, available from Novagen, which showed expression of anabout 55-kDa protein. The protein product was purified by liquidchromatography using a HiTrap™ chelating column charged with NiCl₂,available from Pharmacia, and was shown to contain the His tag of thevector when subjected to automated protein sequencing by Edmandegradation.

C. Northern Blot Analysis

A Northern Blot analysis was conducted as follows to determine whetherallantoinase is expressed exclusively in HMT tissues. HMT tissues weredissected from 1000 adult cat blood-fed C. felis having a male to femaleratio of 1:4. Total RNA was separately extracted from HMT tissues andthe HMT-less carcasses that resulted from these dissections as follows.The tissues were frozen at −80° C., ground into a powder with a mortarand pestle, and the powders were equally divided into four 2-mleppendorf tubes each containing 1 ml of lysis buffer. The lysis buffercontained 4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 3%sarcosyl, 0.5 M 2-mercaptoethanol, 0.1% antifoam, and 1 mMaurintricarboxylic acid, all available from Sigma Chemical Corporation,St. Louis, Mo. After mixing, the tubes were spun at 14,000 rpm for 2minutes and the supernatants were transferred to separate 2 ml eppendorftubes containing 250 μl of phenol, available from Aldrich, Milwaukee,Wis. After mixing, the tubes were spun at 14,000 rpm for 5 minutes andthe supernatants were transferred to new 2-ml tubes. This process wasrepeated 3 times until no proteinaceous matter was visible at thephenol/lysis buffer interface, then 250 μl of chloroform was added toeach tube and the contents mixed and spun at 14,000 rpm for 5 minutesfollowed by transferring the supernatant to a new tube. A volume ofisopropanol equal to the volume of the supernatant was added to eachtube and the tubes placed on ice for 5 minutes. The tubes were then spunat 14,000 rpm at room temperature for 15 minutes, the supernatants wereremoved and discarded and the remaining RNA pellets were washed with 70%ethanol and dried. The RNA pellets were resuspended in 100 μl of TE (10mM Tris, 1 mM ethylenediaminetetraacetic acid (EDTA)). The quantity ofRNA in each tube was then determined using a spectrophotometer.

Approximately 10 μg of each RNA was added to separate tubes containing18.75 μl of loading buffer, which consists of 50% formamide, 16%formaldehyde, 17% water, 7% glycerol, 1×MOPS buffer (a 1:20 dilution of0.4 M 93-[N-morpholino]propanesulfonic acid (MOPS), 0.1 M sodiumacetate, and 20 mM EDTA), 10 μl ethidium bromide, and 10 μl bromophenolblue dye, all available from Sigma. The tubes were heated to 95° C. for2 minutes then placed on ice. The RNA samples were separated by gelelectrophoresis on a 1.5% agarose gel with 3.2% formaldehyde and 1×MOPSbuffer; the gel was then soaked in water for 30 minutes prior totransfer to remove excess formaldehyde. The gel was then transferredusing standard techniques, described by Sambrook et al., ibid, with10×SSPE as the transfer buffer onto Nytran® nylon membrane, availablefrom Schleicher and Schuell Inc., Keene, N.H. The membrane was UVcross-linked using the Stratalinker®, available from Stratagene, thenprehybridized at 42° C. in 50% formamide, 5×SSPE, 1.2% SDS, 5×Denhardt'sreagent, 2.5 mM EDTA, and 100 μg/ml salmon sperm DNA. A probe comprisingthe allantoinase EST nucleic acid molecule, nCfALN₆₈₂ was labeled withα-³²P-ATP using a DNA labeling kit, available from Amersham and added tothe buffer at a concentration of approximately 1×10⁶ cpm/ml, and allowedto hybridize for 18 hours at 42° C. The blot was then washed as follows:10 minutes at 42° C. in 4×SSPE and 1% SDS; 10 minutes at 42° C. in2×SSPE and 1% SDS; 10 minutes at 42° C. with 0.5×SSPE and 0.5×SDS; and10 minutes at 42° C. with 0.25×SSPE and 0.25% SDS. The blot was thenexposed to film for I hour, and the film was developed using standardprocedures. Analysis of the developed film revealed that allantoinasemRNA was present in HMT tissues but was not present in non-HMT tissues.

Northern Blot analysis was also conducted to determine whetherallantoinase mRNA is expressed only in certain stages of the flea lifecycle and whether allantoinase mRNA expression is influenced by feeding.Total RNA was extracted as described above from 1000 fleas at each ofthe following flea life stages: eggs; first instar larvae; third instarlarvae; wandering larvae; and pupae; and from 1000 adult fleas under thefollowing feeding conditions: unfed, fed on cat blood for 15 minutes,fed on cat blood for 2 hours, fed on cat blood for 8 hours, and fed oncat blood for 24 hours.

Each RNA sample was separated by gel electrophoresis, transferred tonylon membrane and hybridized with α-³²P-ATP labeled nCfALN₆₈₂ probe asdescribed above. Analysis of the developed film revealed thatallantoinase mRNA was expressed in all adult fleas tested regardless offeeding conditions and was expressed by all life stages except for eggsand pupae, the two life stages which do not feed or excrete urine.

D. Production of Affinity-Purified Antibodies

Affinity-purified rabbit antibodies against a C. felis allantoinaseprotein of the present invention were generated as follows. Rabbits wereimmunized three times with 50 μg each of recombinant C. felisallantoinase protein PCfALN₃₇₄ that was produced as described above andpurified by nickel chelating chromatography. The primary immunizationwas conducted with protein mixed 1:1 in Freund's complete adjuvant andbooster immunizations were conducted with protein mixed 1:1 in Freund'sincomplete adjuvant. Ten milliliters of rabbit sera were collected eachweek for 12 weeks and antibodies specific to contaminating E. coliproteins were removed by incubation with a piece of nitrocellulose thathad been blocked in E. coli cell lysate. The decontaminated rabbit serumwas then affinity purified using Sepharose 4B beads coupled to purifiedrecombinant C. felis allantoinase protein, PCfALN₃₇₄ to produce rabbitantibodies against C. felis allantoinase protein PCfALN₃₇₄ (also denotedα-C. felis allantoinase antibodies).

A Western blot analysis was conducted using the α-C. felis allantoinaseantibodies to detect native flea allantoinase protein in flea proteinextracts. Nine μg total protein from flea protein extract prepared asdescribed in section E were separated by SDS-PAGE and transferred to anitrocellulose membrane using standard methods. A 1:1000 dilution of theα-C. felis allantoinase antibodies was used as the primary antibody, anda 1:10,000 dilution of alkaline phosphatase conjugated goat α-rabbitIgG, available from Kirkegaard & Perry Laboratories, Gaithersburg, Md.,was used as the secondary antibody. The blot detected two distinctbands. The size of each band was approximated using two differentprotein markers, the SeeBlue™ standard and the Mark12™ standard, eachavailable from Novex, San Diego, Calif. Size approximation based uponthe SeeBlue™ standard indicated that the bands were 62 and 65 kDa; andsize approximation based upon the Mark12™ standard indicated that thebands were 53 and 55 kDa. These bands are hereinafter referred to as the“53 kDa” and the “55 kDa” bands, respectively.

E. Allantoinase Activity Assay

Allantoinase activity of C. felis lysates was assessed by the ability ofthe lysate to convert allantoin to allantoic acid as follows. Fleaprotein was extracted from 5.6 grams of unfed adult fleas by grindingfleas into powder using a chilled mortar and pestle followed bysuspension in 1×PBS. The mixture was frozen and thawed three times inliquid nitrogen and a 37° C. water bath. The mixture was then sonicatedfor 60 seconds and centrifuged at 25,000×G for 20 minutes. Thesupernatant was collected.

The protein concentration of the flea protein extract was adjusted to0.925 mg/ml with the addition of 1×PBS. Ten μl of this preparation wasincubated with 44 μl of 45 mM allantoin and 6 μl of Hepes buffer at roomtemperature for 20 minutes. The reaction was stopped by the addition of10 μl of 0.25 N HCl and the mixture was heated to 95° C. for 5 minutesto convert the allantoic acid reaction product into glyoxylic acid andurea. Ten μl of 5 mM 2,4-dinitrophenylhydrazine was added as thecalorimetric indicator and the reaction was incubated for 15 minutes atroom temperature. One-hundred and twenty μl of 0.5 M sodium phosphatewas added and the mixture was incubated at room temperature for 15minutes. The presence of allantoinase in the flea protein extract wasindicated by the conversion of the indicator by glyoxylic acid intodinitrophenylhydrazone, which, following the addition of base had anorange-brown color the absorbance of which was read at 450 nm. Usingthis method, unfed adult fleas and fleas fed for 24 hours on blood wereshown to have similar allantoinase activity. Commercially availablepeanut allantoinase, available from Sigma, St. Louis, Mo., was used as apositive control and also showed activity using this method.

Acetohydroxamic acid (AHA) a reported allantoinase inhibitor, was testedin the assay. Six μl of AHA, available from Sigma, St. Louis, Mo., wasdiluted in dimethyl sulfoxide (DMSO) and added to 44 μl of 45 mMallantoin and 10 μl of flea protein extract and the mixture wasincubated for 30 minutes at room temperature. An allantoinase activityassay was performed as described above. AHA inhibited flea allantoinaseactivity in a dose-dependent manner. The IC₅₀ and IC₉₀ values weremeasured and determined to be 20 mM and 60 mM, respectively. AHA wasalso tested for the ability to inhibit peanut allantoinase activity,with nearly identical results.

F. Partial Purification of Native Protein

A partially purified active native C. felis allantoinase protein wasproduced as follows. About 9 milligrams of C. felis lysate were passedthrough a gel filtration column using 1×PBS as the buffer. A Westernblot was performed on elution fractions 1-19 using α-C. felisallantoinase antibodies prepared as described above. The blot showed a55 kDa band in fractions 11-17 and a 53 kDa band in fractions 14-19 thatwere recognized by the antibodies. Activity assays were performed asdescribed above on all elution fractions and only fractions 11-19 showedallantoinase activity.

Fractions having as their major component the 53 kDa band, i.e.fractions 16-19, were combined and further purified by anion exchangechromatography. A Western blot performed on fractions from thatchromatography using α-C. felis allantoinase antibodies detected a 53kDa band in fractions 6-9. All fractions were tested for activity andonly fractions 6-9 were shown to have allantoinase activity. Fractions6-9 were combined and further purified by affinity column chromatographyusing an affinity column constructed by coupling approximately 1 mg ofα-C. felis allantoinase antibodies to Sepharose 4B beads. Elutionfractions 1-5 showed activity, with the greatest allantoinase activityin the first fraction; fractions which eluted after fraction 5 showed noactivity. The first fraction was subjected to SDS-PAGE followed bysilver staining which showed a nearly pure 53 kDa protein band.Fractions 1-5 were combined and concentrated using a Centricon Plus-20concentrator and subjected to Western blot analysis using α-C. felisallantoinase antibodies which revealed a single 53 kDa band.

Elution fractions from the gel filtration column having as their majorcomponent the 55 kDa band, i.e. fractions 12-15, were combined andfurther purified by cation exchange chromatography. A Western blotperformed on fractions 3-6 from that chromatography using α-C. felisallantoinase antibodies detected a 55 kDa band. Fractions 3-6 weretested and shown to have allantoinase activity. Fractions which did notcontain the 55 kDa band did not exhibit allantoinase activity.

G. Protein Localization Study

Protein localization in thin sections of fleas was conducted usingimmunohistochemistry as follows. Unfed adult fleas were fixed inparaformaldehyde, imbedded with paraffin, thin sectioned and fixed ontoglass cover slips using standard techniques. A 1:1000 dilution of serumcontaining rabbit α-C. felis allantoinase antibodies, produced asdescribed above, was used in combination with a 1:10,000 dilution ofanti-rabbit secondary antibody, from a StrAviGen™ Super SensitiveImmunodetection System and substrate from an AEC Substrate Pack, eachavailable from Bio Genex, San Ramon, Calif. for localization of thenative protein within the thin sections. This localization procedurerevealed that allantoinase protein is present in the Malpighian tubules,hindgut and rectum of adult fleas, and is not present in other tissuesof the flea. The same procedure was conducted using normal rabbit seradiluted 1:500 as a negative control and no staining of tissues wasobserved.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims:

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 15 <210> SEQ ID NO 1 <211> LENGTH: 2035<212> TYPE: DNA <213> ORGANISM: Ctenocephalides felis <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (152)..(1600) <400> SEQUENCE: 1aacataataa taacttaata aaattttgtg atcagatttc taatatccag aa#caaagcca     60gtaattataa gaaccaagcc tatttcatgt gaaggttact tctccacagt at#tattatct    120 atctcaagaa gtaatctatt actgaatcaa a atg aaa agc agt #acc tgt att       172                    #                 Met# Lys Ser Ser Thr Cys Ile                    #                  # 1               5 ttt ctt ctg gtc att atg ctg aat tgc aag aa#c ctt gtt aat gct gcg      220Phe Leu Leu Val Ile Met Leu Asn Cys Lys As #n Leu Val Asn Ala Ala         10          #         15          #         20tgc acc aac aac gcg cct cca atg aag ata tt#c cgt agc cga aga gtt      268Cys Thr Asn Asn Ala Pro Pro Met Lys Ile Ph #e Arg Ser Arg Arg Val     25              #     30              #     35ctt ctc ggt gat ggt act gaa aga gat gct gg#c att gta gtt gat tcc      316Leu Leu Gly Asp Gly Thr Glu Arg Asp Ala Gl #y Ile Val Val Asp Ser 40                  # 45                  # 50                  # 55tcc gga aga ata aaa agt ata att tca gga ga#a gaa gtg gaa agg ata      364Ser Gly Arg Ile Lys Ser Ile Ile Ser Gly Gl #u Glu Val Glu Arg Ile                 60  #                 65  #                 70gct aac gaa act aaa gtt gag gtg ttg gac ta#c ggt caa ttt tca ata      412Ala Asn Glu Thr Lys Val Glu Val Leu Asp Ty #r Gly Gln Phe Ser Ile             75      #             80      #             85tgg cca ggt gtg ata gac tct cat gtg cac gt#c aac gaa cca gga aga      460Trp Pro Gly Val Ile Asp Ser His Val His Va #l Asn Glu Pro Gly Arg         90          #         95          #        100gaa tcc tgg gaa gga tac acc aca gct act aa#a gca gca gct tgg ggc      508Glu Ser Trp Glu Gly Tyr Thr Thr Ala Thr Ly #s Ala Ala Ala Trp Gly    105               #   110               #   115ggg att acc aca ata gta gac atg cct ttg aa#t tcc atc cca cct aca      556Gly Ile Thr Thr Ile Val Asp Met Pro Leu As #n Ser Ile Pro Pro Thr120                 1 #25                 1 #30                 1 #35act act gta gag aat ttg aga aca aaa gtg aa#t tca gcc tgt ggt aaa      604Thr Thr Val Glu Asn Leu Arg Thr Lys Val As #n Ser Ala Cys Gly Lys                140   #               145   #               150acg cat gtt gat gtc gct ttc tgg gga ggc gt#g att cct ggc aat gcg      652Thr His Val Asp Val Ala Phe Trp Gly Gly Va #l Ile Pro Gly Asn Ala            155       #           160       #           165cac gaa ttg ttg cca ctt atc aac gcc gga gt#a aga gga ttc aaa tgt      700His Glu Leu Leu Pro Leu Ile Asn Ala Gly Va #l Arg Gly Phe Lys Cys        170           #       175           #       180ttt aca agt gaa agt ggt gtc gat gag ttt cc#a cag gtt act aaa aat      748Phe Thr Ser Glu Ser Gly Val Asp Glu Phe Pr #o Gln Val Thr Lys Asn    185               #   190               #   195gat ctg gaa atg gct cta aaa gag ctc cag aa#a gca aat tcc gta ctt      796Asp Leu Glu Met Ala Leu Lys Glu Leu Gln Ly #s Ala Asn Ser Val Leu200                 2 #05                 2 #10                 2 #15ctg tac cat gcc gaa tta ccc gct cct caa ga#a aat gtt aca agc aat      844Leu Tyr His Ala Glu Leu Pro Ala Pro Gln Gl #u Asn Val Thr Ser Asn                220   #               225   #               230gaa act gaa aag tac atg act tac ctg aaa ac#a cga cct cca agt atg      892Glu Thr Glu Lys Tyr Met Thr Tyr Leu Lys Th #r Arg Pro Pro Ser Met            235       #           240       #           245gaa gta aat gct att gat atg att ata gac ct#c aca aaa aaa tat aaa      940Glu Val Asn Ala Ile Asp Met Ile Ile Asp Le #u Thr Lys Lys Tyr Lys        250           #       255           #       260gtt agg tct cac ata gtg cat cta tca gca gc#a ggt gct tta ccg caa      988Val Arg Ser His Ile Val His Leu Ser Ala Al #a Gly Ala Leu Pro Gln    265               #   270               #   275ttg aaa aaa gcg cgc tca gag aac gtt cca ct#t tcg att gaa act tgt     1036Leu Lys Lys Ala Arg Ser Glu Asn Val Pro Le #u Ser Ile Glu Thr Cys280                 2 #85                 2 #90                 2 #95cat cat tac tta acc ttt gct gct gaa gat gt#t cca gat gga cat act     1084His His Tyr Leu Thr Phe Ala Ala Glu Asp Va #l Pro Asp Gly His Thr                300   #               305   #               310gaa tac aaa tgc gct cca cca att aga gaa ga#a agt aat caa gaa aaa     1132Glu Tyr Lys Cys Ala Pro Pro Ile Arg Glu Gl #u Ser Asn Gln Glu Lys            315       #           320       #           325tta tgg caa gct ttg gaa aac aga gat att ga#t atg gta gtc agt gat     1180Leu Trp Gln Ala Leu Glu Asn Arg Asp Ile As #p Met Val Val Ser Asp        330           #       335           #       340cat tct cca tca cct gct gca ctg aaa ggc ct#g tgc aat ggt tgt cat     1228His Ser Pro Ser Pro Ala Ala Leu Lys Gly Le #u Cys Asn Gly Cys His    345               #   350               #   355cct gat ttc cta aaa gct tgg ggt gga att gc#t ggt atg cag ttt gga     1276Pro Asp Phe Leu Lys Ala Trp Gly Gly Ile Al #a Gly Met Gln Phe Gly360                 3 #65                 3 #70                 3 #75tta tct tta ata agg acc ggt gct tct aaa ag#a ggc ttt aaa gct cat     1324Leu Ser Leu Ile Arg Thr Gly Ala Ser Lys Ar #g Gly Phe Lys Ala His                380   #               385   #               390gat gta tct cgt tta tta tct gcg gga cct gc#g aaa tta act gga ctg     1372Asp Val Ser Arg Leu Leu Ser Ala Gly Pro Al #a Lys Leu Thr Gly Leu            395       #           400       #           405gat ggc ata aaa gga caa atc aaa gaa ggc tt#g gat gct gat tta gta     1420Asp Gly Ile Lys Gly Gln Ile Lys Glu Gly Le #u Asp Ala Asp Leu Val        410           #       415           #       420att tgg gat cct gag gaa gaa ttt aag gtc ac#t aaa gac ata atc caa     1468Ile Trp Asp Pro Glu Glu Glu Phe Lys Val Th #r Lys Asp Ile Ile Gln    425               #   430               #   435cac aag aat aaa gaa aca cca tac tta gga at#g acg ttg aag ggc aaa     1516His Lys Asn Lys Glu Thr Pro Tyr Leu Gly Me #t Thr Leu Lys Gly Lys440                 4 #45                 4 #50                 4 #55gtt cat gca act gtt gta cga gga gac ttt gt#t tac cgt aat gga caa     1564Val His Ala Thr Val Val Arg Gly Asp Phe Va #l Tyr Arg Asn Gly Gln                460   #               465   #               470cca ttc gaa att cca aaa gga aat tta ctt at#t gaa tgattaaatg          1610Pro Phe Glu Ile Pro Lys Gly Asn Leu Leu Il #e Glu             475      #           480taatagatta atcaaatttt agatgattaa aattgtttta ttactacaat ag#caacctct   1670gcctgaaaat taaccgaaca aacttctaac atccttatta atgtatagat tt#tgaataat   1730aacatagaaa ttatactatt tttttgatga ctctaataaa aaaaatgtat aa#atggccat   1790gcctgatata tttttgataa ccttaatgaa aaaatgttta aatggccatg tc#tgaaaaga   1850tttctatgtg tatttttttg ttaacatttt attgttgaat ggataaaaga ta#aatacaat   1910tttataagct gtttggataa attaattttg aataaatcca taatcataga at#atgttaag   1970tagcaaatta aaatatggac cacaaaccac aaaatgtata cgaaatataa ct#tatatgat   2030 atatg                  #                  #                   #          2035 <210> SEQ ID NO 2 <211> LENGTH: 483<212> TYPE: PRT <213> ORGANISM: Ctenocephalides felis <400> SEQUENCE: 2Met Lys Ser Ser Thr Cys Ile Phe Leu Leu Va #l Ile Met Leu Asn Cys  1               5  #                 10  #                 15Lys Asn Leu Val Asn Ala Ala Cys Thr Asn As #n Ala Pro Pro Met Lys             20      #             25      #             30Ile Phe Arg Ser Arg Arg Val Leu Leu Gly As #p Gly Thr Glu Arg Asp         35          #         40          #         45Ala Gly Ile Val Val Asp Ser Ser Gly Arg Il #e Lys Ser Ile Ile Ser     50              #     55              #     60Gly Glu Glu Val Glu Arg Ile Ala Asn Glu Th #r Lys Val Glu Val Leu 65                  # 70                  # 75                  # 80Asp Tyr Gly Gln Phe Ser Ile Trp Pro Gly Va #l Ile Asp Ser His Val                 85  #                 90  #                 95His Val Asn Glu Pro Gly Arg Glu Ser Trp Gl #u Gly Tyr Thr Thr Ala            100       #           105       #           110Thr Lys Ala Ala Ala Trp Gly Gly Ile Thr Th #r Ile Val Asp Met Pro        115           #       120           #       125Leu Asn Ser Ile Pro Pro Thr Thr Thr Val Gl #u Asn Leu Arg Thr Lys    130               #   135               #   140Val Asn Ser Ala Cys Gly Lys Thr His Val As #p Val Ala Phe Trp Gly145                 1 #50                 1 #55                 1 #60Gly Val Ile Pro Gly Asn Ala His Glu Leu Le #u Pro Leu Ile Asn Ala                165   #               170   #               175Gly Val Arg Gly Phe Lys Cys Phe Thr Ser Gl #u Ser Gly Val Asp Glu            180       #           185       #           190Phe Pro Gln Val Thr Lys Asn Asp Leu Glu Me #t Ala Leu Lys Glu Leu        195           #       200           #       205Gln Lys Ala Asn Ser Val Leu Leu Tyr His Al #a Glu Leu Pro Ala Pro    210               #   215               #   220Gln Glu Asn Val Thr Ser Asn Glu Thr Glu Ly #s Tyr Met Thr Tyr Leu225                 2 #30                 2 #35                 2 #40Lys Thr Arg Pro Pro Ser Met Glu Val Asn Al #a Ile Asp Met Ile Ile                245   #               250   #               255Asp Leu Thr Lys Lys Tyr Lys Val Arg Ser Hi #s Ile Val His Leu Ser            260       #           265       #           270Ala Ala Gly Ala Leu Pro Gln Leu Lys Lys Al #a Arg Ser Glu Asn Val        275           #       280           #       285Pro Leu Ser Ile Glu Thr Cys His His Tyr Le #u Thr Phe Ala Ala Glu    290               #   295               #   300Asp Val Pro Asp Gly His Thr Glu Tyr Lys Cy #s Ala Pro Pro Ile Arg305                 3 #10                 3 #15                 3 #20Glu Glu Ser Asn Gln Glu Lys Leu Trp Gln Al #a Leu Glu Asn Arg Asp                325   #               330   #               335Ile Asp Met Val Val Ser Asp His Ser Pro Se #r Pro Ala Ala Leu Lys            340       #           345       #           350Gly Leu Cys Asn Gly Cys His Pro Asp Phe Le #u Lys Ala Trp Gly Gly        355           #       360           #       365Ile Ala Gly Met Gln Phe Gly Leu Ser Leu Il #e Arg Thr Gly Ala Ser    370               #   375               #   380Lys Arg Gly Phe Lys Ala His Asp Val Ser Ar #g Leu Leu Ser Ala Gly385                 3 #90                 3 #95                 4 #00Pro Ala Lys Leu Thr Gly Leu Asp Gly Ile Ly #s Gly Gln Ile Lys Glu                405   #               410   #               415Gly Leu Asp Ala Asp Leu Val Ile Trp Asp Pr #o Glu Glu Glu Phe Lys            420       #           425       #           430Val Thr Lys Asp Ile Ile Gln His Lys Asn Ly #s Glu Thr Pro Tyr Leu        435           #       440           #       445Gly Met Thr Leu Lys Gly Lys Val His Ala Th #r Val Val Arg Gly Asp    450               #   455               #   460Phe Val Tyr Arg Asn Gly Gln Pro Phe Glu Il #e Pro Lys Gly Asn Leu465                 4 #70                 4 #75                 4 #80Leu Ile Glu <210> SEQ ID NO 3 <211> LENGTH: 2035 <212> TYPE: DNA<213> ORGANISM: Ctenocephalides felis <400> SEQUENCE: 3catatatcat ataagttata tttcgtatac attttgtggt ttgtggtcca ta#ttttaatt     60tgctacttaa catattctat gattatggat ttattcaaaa ttaatttatc ca#aacagctt    120ataaaattgt atttatcttt tatccattca acaataaaat gttaacaaaa aa#atacacat    180agaaatcttt tcagacatgg ccatttaaac attttttcat taaggttatc aa#aaatatat    240caggcatggc catttataca ttttttttat tagagtcatc aaaaaaatag ta#taatttct    300atgttattat tcaaaatcta tacattaata aggatgttag aagtttgttc gg#ttaatttt    360caggcagagg ttgctattgt agtaataaaa caattttaat catctaaaat tt#gattaatc    420tattacattt aatcattcaa taagtaaatt tccttttgga atttcgaatg gt#tgtccatt    480acggtaaaca aagtctcctc gtacaacagt tgcatgaact ttgcccttca ac#gtcattcc    540taagtatggt gtttctttat tcttgtgttg gattatgtct ttagtgacct ta#aattcttc    600ctcaggatcc caaattacta aatcagcatc caagccttct ttgatttgtc ct#tttatgcc    660atccagtcca gttaatttcg caggtcccgc agataataaa cgagatacat ca#tgagcttt    720aaagcctctt ttagaagcac cggtccttat taaagataat ccaaactgca ta#ccagcaat    780tccaccccaa gcttttagga aatcaggatg acaaccattg cacaggcctt tc#agtgcagc    840aggtgatgga gaatgatcac tgactaccat atcaatatct ctgttttcca aa#gcttgcca    900taatttttct tgattacttt cttctctaat tggtggagcg catttgtatt ca#gtatgtcc    960atctggaaca tcttcagcag caaaggttaa gtaatgatga caagtttcaa tc#gaaagtgg   1020aacgttctct gagcgcgctt ttttcaattg cggtaaagca cctgctgctg at#agatgcac   1080tatgtgagac ctaactttat atttttttgt gaggtctata atcatatcaa ta#gcatttac   1140ttccatactt ggaggtcgtg ttttcaggta agtcatgtac ttttcagttt ca#ttgcttgt   1200aacattttct tgaggagcgg gtaattcggc atggtacaga agtacggaat tt#gctttctg   1260gagctctttt agagccattt ccagatcatt tttagtaacc tgtggaaact ca#tcgacacc   1320actttcactt gtaaaacatt tgaatcctct tactccggcg ttgataagtg gc#aacaattc   1380gtgcgcattg ccaggaatca cgcctcccca gaaagcgaca tcaacatgcg tt#ttaccaca   1440ggctgaattc acttttgttc tcaaattctc tacagtagtt gtaggtggga tg#gaattcaa   1500aggcatgtct actattgtgg taatcccgcc ccaagctgct gctttagtag ct#gtggtgta   1560tccttcccag gattctcttc ctggttcgtt gacgtgcaca tgagagtcta tc#acacctgg   1620ccatattgaa aattgaccgt agtccaacac ctcaacttta gtttcgttag ct#atcctttc   1680cacttcttct cctgaaatta tactttttat tcttccggag gaatcaacta ca#atgccagc   1740atctctttca gtaccatcac cgagaagaac tcttcggcta cggaatatct tc#attggagg   1800cgcgttgttg gtgcacgcag cattaacaag gttcttgcaa ttcagcataa tg#accagaag   1860aaaaatacag gtactgcttt tcattttgat tcagtaatag attacttctt ga#gatagata   1920ataatactgt ggagaagtaa ccttcacatg aaataggctt ggttcttata at#tactggct   1980ttgttctgga tattagaaat ctgatcacaa aattttatta agttattatt at#gtt        2035 <210> SEQ ID NO 4 <211> LENGTH: 1449 <212> TYPE: DNA<213> ORGANISM: Ctenocephalides felis <400> SEQUENCE: 4atgaaaagca gtacctgtat ttttcttctg gtcattatgc tgaattgcaa ga#accttgtt     60aatgctgcgt gcaccaacaa cgcgcctcca atgaagatat tccgtagccg aa#gagttctt    120ctcggtgatg gtactgaaag agatgctggc attgtagttg attcctccgg aa#gaataaaa    180agtataattt caggagaaga agtggaaagg atagctaacg aaactaaagt tg#aggtgttg    240gactacggtc aattttcaat atggccaggt gtgatagact ctcatgtgca cg#tcaacgaa    300ccaggaagag aatcctggga aggatacacc acagctacta aagcagcagc tt#ggggcggg    360attaccacaa tagtagacat gcctttgaat tccatcccac ctacaactac tg#tagagaat    420ttgagaacaa aagtgaattc agcctgtggt aaaacgcatg ttgatgtcgc tt#tctgggga    480ggcgtgattc ctggcaatgc gcacgaattg ttgccactta tcaacgccgg ag#taagagga    540ttcaaatgtt ttacaagtga aagtggtgtc gatgagtttc cacaggttac ta#aaaatgat    600ctggaaatgg ctctaaaaga gctccagaaa gcaaattccg tacttctgta cc#atgccgaa    660ttacccgctc ctcaagaaaa tgttacaagc aatgaaactg aaaagtacat ga#cttacctg    720aaaacacgac ctccaagtat ggaagtaaat gctattgata tgattataga cc#tcacaaaa    780aaatataaag ttaggtctca catagtgcat ctatcagcag caggtgcttt ac#cgcaattg    840aaaaaagcgc gctcagagaa cgttccactt tcgattgaaa cttgtcatca tt#acttaacc    900tttgctgctg aagatgttcc agatggacat actgaataca aatgcgctcc ac#caattaga    960gaagaaagta atcaagaaaa attatggcaa gctttggaaa acagagatat tg#atatggta   1020gtcagtgatc attctccatc acctgctgca ctgaaaggcc tgtgcaatgg tt#gtcatcct   1080gatttcctaa aagcttgggg tggaattgct ggtatgcagt ttggattatc tt#taataagg   1140accggtgctt ctaaaagagg ctttaaagct catgatgtat ctcgtttatt at#ctgcggga   1200cctgcgaaat taactggact ggatggcata aaaggacaaa tcaaagaagg ct#tggatgct   1260gatttagtaa tttgggatcc tgaggaagaa tttaaggtca ctaaagacat aa#tccaacac   1320aagaataaag aaacaccata cttaggaatg acgttgaagg gcaaagttca tg#caactgtt   1380gtacgaggag actttgttta ccgtaatgga caaccattcg aaattccaaa ag#gaaattta   1440 cttattgaa                 #                  #                   #       1449 <210> SEQ ID NO 5 <211> LENGTH: 1449<212> TYPE: DNA <213> ORGANISM: Ctenocephalides felis <400> SEQUENCE: 5ttcaataagt aaatttcctt ttggaatttc gaatggttgt ccattacggt aa#acaaagtc     60tcctcgtaca acagttgcat gaactttgcc cttcaacgtc attcctaagt at#ggtgtttc    120tttattcttg tgttggatta tgtctttagt gaccttaaat tcttcctcag ga#tcccaaat    180tactaaatca gcatccaagc cttctttgat ttgtcctttt atgccatcca gt#ccagttaa    240tttcgcaggt cccgcagata ataaacgaga tacatcatga gctttaaagc ct#cttttaga    300agcaccggtc cttattaaag ataatccaaa ctgcatacca gcaattccac cc#caagcttt    360taggaaatca ggatgacaac cattgcacag gcctttcagt gcagcaggtg at#ggagaatg    420atcactgact accatatcaa tatctctgtt ttccaaagct tgccataatt tt#tcttgatt    480actttcttct ctaattggtg gagcgcattt gtattcagta tgtccatctg ga#acatcttc    540agcagcaaag gttaagtaat gatgacaagt ttcaatcgaa agtggaacgt tc#tctgagcg    600cgcttttttc aattgcggta aagcacctgc tgctgataga tgcactatgt ga#gacctaac    660tttatatttt tttgtgaggt ctataatcat atcaatagca tttacttcca ta#cttggagg    720tcgtgttttc aggtaagtca tgtacttttc agtttcattg cttgtaacat tt#tcttgagg    780agcgggtaat tcggcatggt acagaagtac ggaatttgct ttctggagct ct#tttagagc    840catttccaga tcatttttag taacctgtgg aaactcatcg acaccacttt ca#cttgtaaa    900acatttgaat cctcttactc cggcgttgat aagtggcaac aattcgtgcg ca#ttgccagg    960aatcacgcct ccccagaaag cgacatcaac atgcgtttta ccacaggctg aa#ttcacttt   1020tgttctcaaa ttctctacag tagttgtagg tgggatggaa ttcaaaggca tg#tctactat   1080tgtggtaatc ccgccccaag ctgctgcttt agtagctgtg gtgtatcctt cc#caggattc   1140tcttcctggt tcgttgacgt gcacatgaga gtctatcaca cctggccata tt#gaaaattg   1200accgtagtcc aacacctcaa ctttagtttc gttagctatc ctttccactt ct#tctcctga   1260aattatactt tttattcttc cggaggaatc aactacaatg ccagcatctc tt#tcagtacc   1320atcaccgaga agaactcttc ggctacggaa tatcttcatt ggaggcgcgt tg#ttggtgca   1380cgcagcatta acaaggttct tgcaattcag cataatgacc agaagaaaaa ta#caggtact   1440 gcttttcat                 #                  #                   #       1449 <210> SEQ ID NO 6 <211> LENGTH: 1383<212> TYPE: DNA <213> ORGANISM: Ctenocephalides felis <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(1383) <400> SEQUENCE: 6gcg tgc acc aac aac gcg cct cca atg aag at #a ttc cgt agc cga aga   48Ala Cys Thr Asn Asn Ala Pro Pro Met Lys Il #e Phe Arg Ser Arg Arg  1               5  #                 10  #                 15gtt ctt ctc ggt gat ggt act gaa aga gat gc #t ggc att gta gtt gat   96Val Leu Leu Gly Asp Gly Thr Glu Arg Asp Al #a Gly Ile Val Val Asp             20      #             25      #             30tcc tcc gga aga ata aaa agt ata att tca gg #a gaa gaa gtg gaa agg   144Ser Ser Gly Arg Ile Lys Ser Ile Ile Ser Gl #y Glu Glu Val Glu Arg         35          #         40          #         45ata gct aac gaa act aaa gtt gag gtg ttg ga #c tac ggt caa ttt tca   192Ile Ala Asn Glu Thr Lys Val Glu Val Leu As #p Tyr Gly Gln Phe Ser     50              #     55              #     60ata tgg cca ggt gtg ata gac tct cat gtg ca #c gtc aac gaa cca gga   240Ile Trp Pro Gly Val Ile Asp Ser His Val Hi #s Val Asn Glu Pro Gly 65                  # 70                  # 75                  # 80aga gaa tcc tgg gaa gga tac acc aca gct ac #t aaa gca gca gct tgg   288Arg Glu Ser Trp Glu Gly Tyr Thr Thr Ala Th #r Lys Ala Ala Ala Trp                 85  #                 90  #                 95ggc ggg att acc aca ata gta gac atg cct tt #g aat tcc atc cca cct   336Gly Gly Ile Thr Thr Ile Val Asp Met Pro Le #u Asn Ser Ile Pro Pro            100       #           105       #           110aca act act gta gag aat ttg aga aca aaa gt #g aat tca gcc tgt ggt   384Thr Thr Thr Val Glu Asn Leu Arg Thr Lys Va #l Asn Ser Ala Cys Gly        115           #       120           #       125aaa acg cat gtt gat gtc gct ttc tgg gga gg #c gtg att cct ggc aat   432Lys Thr His Val Asp Val Ala Phe Trp Gly Gl #y Val Ile Pro Gly Asn    130               #   135               #   140gcg cac gaa ttg ttg cca ctt atc aac gcc gg #a gta aga gga ttc aaa   480Ala His Glu Leu Leu Pro Leu Ile Asn Ala Gl #y Val Arg Gly Phe Lys145                 1 #50                 1 #55                 1 #60tgt ttt aca agt gaa agt ggt gtc gat gag tt #t cca cag gtt act aaa   528Cys Phe Thr Ser Glu Ser Gly Val Asp Glu Ph #e Pro Gln Val Thr Lys                165   #               170   #               175aat gat ctg gaa atg gct cta aaa gag ctc ca #g aaa gca aat tcc gta   576Asn Asp Leu Glu Met Ala Leu Lys Glu Leu Gl #n Lys Ala Asn Ser Val            180       #           185       #           190ctt ctg tac cat gcc gaa tta ccc gct cct ca #a gaa aat gtt aca agc   624Leu Leu Tyr His Ala Glu Leu Pro Ala Pro Gl #n Glu Asn Val Thr Ser        195           #       200           #       205aat gaa act gaa aag tac atg act tac ctg aa #a aca cga cct cca agt   672Asn Glu Thr Glu Lys Tyr Met Thr Tyr Leu Ly #s Thr Arg Pro Pro Ser    210               #   215               #   220atg gaa gta aat gct att gat atg att ata ga #c ctc aca aaa aaa tat   720Met Glu Val Asn Ala Ile Asp Met Ile Ile As #p Leu Thr Lys Lys Tyr225                 2 #30                 2 #35                 2 #40aaa gtt agg tct cac ata gtg cat cta tca gc #a gca ggt gct tta ccg   768Lys Val Arg Ser His Ile Val His Leu Ser Al #a Ala Gly Ala Leu Pro                245   #               250   #               255caa ttg aaa aaa gcg cgc tca gag aac gtt cc #a ctt tcg att gaa act   816Gln Leu Lys Lys Ala Arg Ser Glu Asn Val Pr #o Leu Ser Ile Glu Thr            260       #           265       #           270tgt cat cat tac tta acc ttt gct gct gaa ga #t gtt cca gat gga cat   864Cys His His Tyr Leu Thr Phe Ala Ala Glu As #p Val Pro Asp Gly His        275           #       280           #       285act gaa tac aaa tgc gct cca cca att aga ga #a gaa agt aat caa gaa   912Thr Glu Tyr Lys Cys Ala Pro Pro Ile Arg Gl #u Glu Ser Asn Gln Glu    290               #   295               #   300aaa tta tgg caa gct ttg gaa aac aga gat at #t gat atg gta gtc agt   960Lys Leu Trp Gln Ala Leu Glu Asn Arg Asp Il #e Asp Met Val Val Ser305                 3 #10                 3 #15                 3 #20gat cat tct cca tca cct gct gca ctg aaa gg #c ctg tgc aat ggt tgt   1008Asp His Ser Pro Ser Pro Ala Ala Leu Lys Gl #y Leu Cys Asn Gly Cys                325   #               330   #               335cat cct gat ttc cta aaa gct tgg ggt gga at #t gct ggt atg cag ttt   1056His Pro Asp Phe Leu Lys Ala Trp Gly Gly Il #e Ala Gly Met Gln Phe            340       #           345       #           350gga tta tct tta ata agg acc ggt gct tct aa #a aga ggc ttt aaa gct   1104Gly Leu Ser Leu Ile Arg Thr Gly Ala Ser Ly #s Arg Gly Phe Lys Ala        355           #       360           #       365cat gat gta tct cgt tta tta tct gcg gga cc #t gcg aaa tta act gga   1152His Asp Val Ser Arg Leu Leu Ser Ala Gly Pr #o Ala Lys Leu Thr Gly    370               #   375               #   380ctg gat ggc ata aaa gga caa atc aaa gaa gg #c ttg gat gct gat tta   1200Leu Asp Gly Ile Lys Gly Gln Ile Lys Glu Gl #y Leu Asp Ala Asp Leu385                 3 #90                 3 #95                 4 #00gta att tgg gat cct gag gaa gaa ttt aag gt #c act aaa gac ata atc   1248Val Ile Trp Asp Pro Glu Glu Glu Phe Lys Va #l Thr Lys Asp Ile Ile                405   #               410   #               415caa cac aag aat aaa gaa aca cca tac tta gg #a atg acg ttg aag ggc   1296Gln His Lys Asn Lys Glu Thr Pro Tyr Leu Gl #y Met Thr Leu Lys Gly            420       #           425       #           430aaa gtt cat gca act gtt gta cga gga gac tt #t gtt tac cgt aat gga   1344Lys Val His Ala Thr Val Val Arg Gly Asp Ph #e Val Tyr Arg Asn Gly        435           #       440           #       445caa cca ttc gaa att cca aaa gga aat tta ct #t att gaa              # 1383 Gln Pro Phe Glu Ile Pro Lys Gly Asn Leu Le #u Ile Glu    450               #   455               #   460 <210> SEQ ID NO 7<211> LENGTH: 461 <212> TYPE: PRT <213> ORGANISM: Ctenocephalides felis<400> SEQUENCE: 7 Ala Cys Thr Asn Asn Ala Pro Pro Met Lys Il#e Phe Arg Ser Arg Arg   1               5  #                 10 #                 15 Val Leu Leu Gly Asp Gly Thr Glu Arg Asp Al#a Gly Ile Val Val Asp              20      #             25     #             30 Ser Ser Gly Arg Ile Lys Ser Ile Ile Ser Gl#y Glu Glu Val Glu Arg          35          #         40         #         45 Ile Ala Asn Glu Thr Lys Val Glu Val Leu As#p Tyr Gly Gln Phe Ser      50              #     55             #     60 Ile Trp Pro Gly Val Ile Asp Ser His Val Hi#s Val Asn Glu Pro Gly  65                  # 70                 # 75                  # 80 Arg Glu Ser Trp Glu Gly Tyr Thr Thr Ala Th#r Lys Ala Ala Ala Trp                  85  #                 90 #                 95 Gly Gly Ile Thr Thr Ile Val Asp Met Pro Le#u Asn Ser Ile Pro Pro             100       #           105      #           110 Thr Thr Thr Val Glu Asn Leu Arg Thr Lys Va#l Asn Ser Ala Cys Gly         115           #       120          #       125 Lys Thr His Val Asp Val Ala Phe Trp Gly Gl#y Val Ile Pro Gly Asn     130               #   135              #   140 Ala His Glu Leu Leu Pro Leu Ile Asn Ala Gl#y Val Arg Gly Phe Lys 145                 1 #50                 1#55                 1 #60 Cys Phe Thr Ser Glu Ser Gly Val Asp Glu Ph#e Pro Gln Val Thr Lys                 165   #               170  #               175 Asn Asp Leu Glu Met Ala Leu Lys Glu Leu Gl#n Lys Ala Asn Ser Val             180       #           185      #           190 Leu Leu Tyr His Ala Glu Leu Pro Ala Pro Gl#n Glu Asn Val Thr Ser         195           #       200          #       205 Asn Glu Thr Glu Lys Tyr Met Thr Tyr Leu Ly#s Thr Arg Pro Pro Ser     210               #   215              #   220 Met Glu Val Asn Ala Ile Asp Met Ile Ile As#p Leu Thr Lys Lys Tyr 225                 2 #30                 2#35                 2 #40 Lys Val Arg Ser His Ile Val His Leu Ser Al#a Ala Gly Ala Leu Pro                 245   #               250  #               255 Gln Leu Lys Lys Ala Arg Ser Glu Asn Val Pr#o Leu Ser Ile Glu Thr             260       #           265      #           270 Cys His His Tyr Leu Thr Phe Ala Ala Glu As#p Val Pro Asp Gly His         275           #       280          #       285 Thr Glu Tyr Lys Cys Ala Pro Pro Ile Arg Gl#u Glu Ser Asn Gln Glu     290               #   295              #   300 Lys Leu Trp Gln Ala Leu Glu Asn Arg Asp Il#e Asp Met Val Val Ser 305                 3 #10                 3#15                 3 #20 Asp His Ser Pro Ser Pro Ala Ala Leu Lys Gl#y Leu Cys Asn Gly Cys                 325   #               330  #               335 His Pro Asp Phe Leu Lys Ala Trp Gly Gly Il#e Ala Gly Met Gln Phe             340       #           345      #           350 Gly Leu Ser Leu Ile Arg Thr Gly Ala Ser Ly#s Arg Gly Phe Lys Ala         355           #       360          #       365 His Asp Val Ser Arg Leu Leu Ser Ala Gly Pr#o Ala Lys Leu Thr Gly     370               #   375              #   380 Leu Asp Gly Ile Lys Gly Gln Ile Lys Glu Gl#y Leu Asp Ala Asp Leu 385                 3 #90                 3#95                 4 #00 Val Ile Trp Asp Pro Glu Glu Glu Phe Lys Va#l Thr Lys Asp Ile Ile                 405   #               410  #               415 Gln His Lys Asn Lys Glu Thr Pro Tyr Leu Gl#y Met Thr Leu Lys Gly             420       #           425      #           430 Lys Val His Ala Thr Val Val Arg Gly Asp Ph#e Val Tyr Arg Asn Gly         435           #       440          #       445 Gln Pro Phe Glu Ile Pro Lys Gly Asn Leu Le #u Ile Glu    450               #   455               #   460 <210> SEQ ID NO 8<211> LENGTH: 1383 <212> TYPE: DNA <213> ORGANISM: Ctenocephalides felis<400> SEQUENCE: 8ttcaataagt aaatttcctt ttggaatttc gaatggttgt ccattacggt aa#acaaagtc     60tcctcgtaca acagttgcat gaactttgcc cttcaacgtc attcctaagt at#ggtgtttc    120tttattcttg tgttggatta tgtctttagt gaccttaaat tcttcctcag ga#tcccaaat    180tactaaatca gcatccaagc cttctttgat ttgtcctttt atgccatcca gt#ccagttaa    240tttcgcaggt cccgcagata ataaacgaga tacatcatga gctttaaagc ct#cttttaga    300agcaccggtc cttattaaag ataatccaaa ctgcatacca gcaattccac cc#caagcttt    360taggaaatca ggatgacaac cattgcacag gcctttcagt gcagcaggtg at#ggagaatg    420atcactgact accatatcaa tatctctgtt ttccaaagct tgccataatt tt#tcttgatt    480actttcttct ctaattggtg gagcgcattt gtattcagta tgtccatctg ga#acatcttc    540agcagcaaag gttaagtaat gatgacaagt ttcaatcgaa agtggaacgt tc#tctgagcg    600cgcttttttc aattgcggta aagcacctgc tgctgataga tgcactatgt ga#gacctaac    660tttatatttt tttgtgaggt ctataatcat atcaatagca tttacttcca ta#cttggagg    720tcgtgttttc aggtaagtca tgtacttttc agtttcattg cttgtaacat tt#tcttgagg    780agcgggtaat tcggcatggt acagaagtac ggaatttgct ttctggagct ct#tttagagc    840catttccaga tcatttttag taacctgtgg aaactcatcg acaccacttt ca#cttgtaaa    900acatttgaat cctcttactc cggcgttgat aagtggcaac aattcgtgcg ca#ttgccagg    960aatcacgcct ccccagaaag cgacatcaac atgcgtttta ccacaggctg aa#ttcacttt   1020tgttctcaaa ttctctacag tagttgtagg tgggatggaa ttcaaaggca tg#tctactat   1080tgtggtaatc ccgccccaag ctgctgcttt agtagctgtg gtgtatcctt cc#caggattc   1140tcttcctggt tcgttgacgt gcacatgaga gtctatcaca cctggccata tt#gaaaattg   1200accgtagtcc aacacctcaa ctttagtttc gttagctatc ctttccactt ct#tctcctga   1260aattatactt tttattcttc cggaggaatc aactacaatg ccagcatctc tt#tcagtacc   1320atcaccgaga agaactcttc ggctacggaa tatcttcatt ggaggcgcgt tg#ttggtgca   1380 cgc                   #                  #                   #           1383 <210> SEQ ID NO 9<211> LENGTH: 1123 <212> TYPE: DNA <213> ORGANISM: Ctenocephalides felis<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (2)..(1123)<400> SEQUENCE: 9 t atg ctg aat tgc aag aac ctt gtt aat gct #gcg tgc acc aac aac gcg     49   Met Leu Asn Cys Lys Asn Leu Val Asn A#la Ala Cys Thr Asn Asn Ala     1               # 5                 # 10                  # 15 cct cca atg aag ata ttc cgt agc cga aga gt#t ctt ctc ggt gat ggt       97Pro Pro Met Lys Ile Phe Arg Ser Arg Arg Va #l Leu Leu Gly Asp Gly             20      #             25      #             30act gaa aga gat gct ggc att gta gtt gat tc#c tcc gga aga ata aaa      145Thr Glu Arg Asp Ala Gly Ile Val Val Asp Se #r Ser Gly Arg Ile Lys         35          #         40          #         45agt ata att tca gga gaa gaa gtg gaa agg at#a gct aac gaa act aaa      193Ser Ile Ile Ser Gly Glu Glu Val Glu Arg Il #e Ala Asn Glu Thr Lys     50              #     55              #     60gtt gag gtg ttg gac tac ggt caa ttt tca at#a tgg cca ggt gtg ata      241Val Glu Val Leu Asp Tyr Gly Gln Phe Ser Il #e Trp Pro Gly Val Ile 65                  # 70                  # 75                  # 80gac tct cat gtg cac gtc aac gaa cca gga ag#a gaa tcc tgg gaa gga      289Asp Ser His Val His Val Asn Glu Pro Gly Ar #g Glu Ser Trp Glu Gly                 85  #                 90  #                 95tac acc aca gct act aaa gca gca gct tgg gg#c ggg att acc aca ata      337Tyr Thr Thr Ala Thr Lys Ala Ala Ala Trp Gl #y Gly Ile Thr Thr Ile            100       #           105       #           110gta gac atg cct ttg aat tcc atc cca cct ac#a act act gta gag aat      385Val Asp Met Pro Leu Asn Ser Ile Pro Pro Th #r Thr Thr Val Glu Asn        115           #       120           #       125ttg aga aca aaa gtg aat tca gcc tgt ggt aa#a acg cat gtt gat gtc      433Leu Arg Thr Lys Val Asn Ser Ala Cys Gly Ly #s Thr His Val Asp Val    130               #   135               #   140gct ttc tgg gga ggc gtg att cct ggc aat gc#g cac gaa ttg ttg cca      481Ala Phe Trp Gly Gly Val Ile Pro Gly Asn Al #a His Glu Leu Leu Pro145                 1 #50                 1 #55                 1 #60ctt atc aac gcc gga gta aga gga ttc aaa tg#t ttt aca agt gaa agt      529Leu Ile Asn Ala Gly Val Arg Gly Phe Lys Cy #s Phe Thr Ser Glu Ser                165   #               170   #               175ggt gtc gat gag ttt cca cag gtt act aaa aa#t gat ctg gaa atg gct      577Gly Val Asp Glu Phe Pro Gln Val Thr Lys As #n Asp Leu Glu Met Ala            180       #           185       #           190cta aaa gag ctc cag aaa gca aat tcc gta ct#t ctg tac cat gcc gaa      625Leu Lys Glu Leu Gln Lys Ala Asn Ser Val Le #u Leu Tyr His Ala Glu        195           #       200           #       205tta ccc gct cct caa gaa aat gtt aca agc aa#t gaa act gaa aag tac      673Leu Pro Ala Pro Gln Glu Asn Val Thr Ser As #n Glu Thr Glu Lys Tyr    210               #   215               #   220atg act tac ctg aaa aca cga cct cca agt at#g gaa gta aat gct att      721Met Thr Tyr Leu Lys Thr Arg Pro Pro Ser Me #t Glu Val Asn Ala Ile225                 2 #30                 2 #35                 2 #40gat atg att ata gac ctc aca aaa aaa tat aa#a gtt agg tct cac ata      769Asp Met Ile Ile Asp Leu Thr Lys Lys Tyr Ly #s Val Arg Ser His Ile                245   #               250   #               255gtg cat cta tca gca gca ggt gct tta ccg ca#a ttg aaa aaa gcg cgc      817Val His Leu Ser Ala Ala Gly Ala Leu Pro Gl #n Leu Lys Lys Ala Arg            260       #           265       #           270tca gag aac gtt cca ctt tcg att gaa act tg#t cat cat tac tta acc      865Ser Glu Asn Val Pro Leu Ser Ile Glu Thr Cy #s His His Tyr Leu Thr        275           #       280           #       285ttt gct gct gaa gat gtt cca gat gga cat ac#t gaa tac aaa tgc gct      913Phe Ala Ala Glu Asp Val Pro Asp Gly His Th #r Glu Tyr Lys Cys Ala    290               #   295               #   300cca cca att aga gaa gaa agt aat caa gaa aa#a tta tgg caa gct ttg      961Pro Pro Ile Arg Glu Glu Ser Asn Gln Glu Ly #s Leu Trp Gln Ala Leu305                 3 #10                 3 #15                 3 #20gaa aac aga gat att gat atg gta gtc agt ga#t cat tct cca tca cct     1009Glu Asn Arg Asp Ile Asp Met Val Val Ser As #p His Ser Pro Ser Pro                325   #               330   #               335gct gca ctg aaa ggc ctg tgc aat ggt tgt ca#t cct gat ttc cta aaa     1057Ala Ala Leu Lys Gly Leu Cys Asn Gly Cys Hi #s Pro Asp Phe Leu Lys            340       #           345       #           350gct tgg ggt gga att gct ggt atg cag ttt gg#a tta tct tta ata agg     1105Ala Trp Gly Gly Ile Ala Gly Met Gln Phe Gl #y Leu Ser Leu Ile Arg        355           #       360           #       365acc ggt gct tct aaa aga          #                   #                  #1123 Thr Gly Ala Ser Lys Arg     370 <210> SEQ ID NO 10<211> LENGTH: 374 <212> TYPE: PRT <213> ORGANISM: Ctenocephalides felis<400> SEQUENCE: 10 Met Leu Asn Cys Lys Asn Leu Val Asn Ala Al#a Cys Thr Asn Asn Ala   1               5  #                 10 #                 15 Pro Pro Met Lys Ile Phe Arg Ser Arg Arg Va#l Leu Leu Gly Asp Gly              20      #             25     #             30 Thr Glu Arg Asp Ala Gly Ile Val Val Asp Se#r Ser Gly Arg Ile Lys          35          #         40         #         45 Ser Ile Ile Ser Gly Glu Glu Val Glu Arg Il#e Ala Asn Glu Thr Lys      50              #     55             #     60 Val Glu Val Leu Asp Tyr Gly Gln Phe Ser Il#e Trp Pro Gly Val Ile  65                  # 70                 # 75                  # 80 Asp Ser His Val His Val Asn Glu Pro Gly Ar#g Glu Ser Trp Glu Gly                  85  #                 90 #                 95 Tyr Thr Thr Ala Thr Lys Ala Ala Ala Trp Gl#y Gly Ile Thr Thr Ile             100       #           105      #           110 Val Asp Met Pro Leu Asn Ser Ile Pro Pro Th#r Thr Thr Val Glu Asn         115           #       120          #       125 Leu Arg Thr Lys Val Asn Ser Ala Cys Gly Ly#s Thr His Val Asp Val     130               #   135              #   140 Ala Phe Trp Gly Gly Val Ile Pro Gly Asn Al#a His Glu Leu Leu Pro 145                 1 #50                 1#55                 1 #60 Leu Ile Asn Ala Gly Val Arg Gly Phe Lys Cy#s Phe Thr Ser Glu Ser                 165   #               170  #               175 Gly Val Asp Glu Phe Pro Gln Val Thr Lys As#n Asp Leu Glu Met Ala             180       #           185      #           190 Leu Lys Glu Leu Gln Lys Ala Asn Ser Val Le#u Leu Tyr His Ala Glu         195           #       200          #       205 Leu Pro Ala Pro Gln Glu Asn Val Thr Ser As#n Glu Thr Glu Lys Tyr     210               #   215              #   220 Met Thr Tyr Leu Lys Thr Arg Pro Pro Ser Me#t Glu Val Asn Ala Ile 225                 2 #30                 2#35                 2 #40 Asp Met Ile Ile Asp Leu Thr Lys Lys Tyr Ly#s Val Arg Ser His Ile                 245   #               250  #               255 Val His Leu Ser Ala Ala Gly Ala Leu Pro Gl#n Leu Lys Lys Ala Arg             260       #           265      #           270 Ser Glu Asn Val Pro Leu Ser Ile Glu Thr Cy#s His His Tyr Leu Thr         275           #       280          #       285 Phe Ala Ala Glu Asp Val Pro Asp Gly His Th#r Glu Tyr Lys Cys Ala     290               #   295              #   300 Pro Pro Ile Arg Glu Glu Ser Asn Gln Glu Ly#s Leu Trp Gln Ala Leu 305                 3 #10                 3#15                 3 #20 Glu Asn Arg Asp Ile Asp Met Val Val Ser As#p His Ser Pro Ser Pro                 325   #               330  #               335 Ala Ala Leu Lys Gly Leu Cys Asn Gly Cys Hi#s Pro Asp Phe Leu Lys             340       #           345      #           350 Ala Trp Gly Gly Ile Ala Gly Met Gln Phe Gl#y Leu Ser Leu Ile Arg         355           #       360          #       365 Thr Gly Ala Ser Lys Arg     370 <210> SEQ ID NO 11<211> LENGTH: 1123 <212> TYPE: DNA <213> ORGANISM: Ctenocephalides felis<400> SEQUENCE: 11tcttttagaa gcaccggtcc ttattaaaga taatccaaac tgcataccag ca#attccacc     60ccaagctttt aggaaatcag gatgacaacc attgcacagg cctttcagtg ca#gcaggtga    120tggagaatga tcactgacta ccatatcaat atctctgttt tccaaagctt gc#cataattt    180ttcttgatta ctttcttctc taattggtgg agcgcatttg tattcagtat gt#ccatctgg    240aacatcttca gcagcaaagg ttaagtaatg atgacaagtt tcaatcgaaa gt#ggaacgtt    300ctctgagcgc gcttttttca attgcggtaa agcacctgct gctgatagat gc#actatgtg    360agacctaact ttatattttt ttgtgaggtc tataatcata tcaatagcat tt#acttccat    420acttggaggt cgtgttttca ggtaagtcat gtacttttca gtttcattgc tt#gtaacatt    480ttcttgagga gcgggtaatt cggcatggta cagaagtacg gaatttgctt tc#tggagctc    540ttttagagcc atttccagat catttttagt aacctgtgga aactcatcga ca#ccactttc    600acttgtaaaa catttgaatc ctcttactcc ggcgttgata agtggcaaca at#tcgtgcgc    660attgccagga atcacgcctc cccagaaagc gacatcaaca tgcgttttac ca#caggctga    720attcactttt gttctcaaat tctctacagt agttgtaggt gggatggaat tc#aaaggcat    780gtctactatt gtggtaatcc cgccccaagc tgctgcttta gtagctgtgg tg#tatccttc    840ccaggattct cttcctggtt cgttgacgtg cacatgagag tctatcacac ct#ggccatat    900tgaaaattga ccgtagtcca acacctcaac tttagtttcg ttagctatcc tt#tccacttc    960ttctcctgaa attatacttt ttattcttcc ggaggaatca actacaatgc ca#gcatctct   1020ttcagtacca tcaccgagaa gaactcttcg gctacggaat atcttcattg ga#ggcgcgtt   1080 gttggtgcac gcagcattaa caaggttctt gcaattcagc ata    #                 112 #3 <210> SEQ ID NO 12 <211> LENGTH: 32<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial #Sequence:  Synthetic       Primer <400> SEQUENCE: 12catgccatgg cgtgcaccaa caacgcgcct cc        #                  #          32 <210> SEQ ID NO 13 <211> LENGTH: 35 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial #Sequence:  Synthetic       Primer <400> SEQUENCE: 13gcggtacctc attcaataag taaatttcct tttgg        #                  #       35 <210> SEQ ID NO 14 <211> LENGTH: 31 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial #Sequence:  Synthetic       Primer <400> SEQUENCE: 14gcggatccta tgctgaattg caagaacctt g         #                  #          31 <210> SEQ ID NO 15 <211> LENGTH: 30 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial #Sequence:  Synthetic       Primer <400> SEQUENCE: 15caggtaccct cttttagaag caccggtccc          #                  #           30

What is claimed is:
 1. An isolated C. felis cDNA or a C. felis RNAnucleic acid molecule selected from the group consisting of (a) a C.felis cDNA or a C. felis RNA that hybridizes to a polynucleotideselected from the group consisting of SEQ ID NO:3 and SEQ ID NO:6, underconditions comprising (1) hybridizing in a solution comprising 1×SSC inthe absence of helix destabilizing compounds, at a temperature of about37° C. and (2) washing in a solution comprising 1×SSC and in the absenceof helix destabilizing compounds, at a temperature of about 47.5° C.,wherein said isolated nucleic acid molecule encodes a protein havingallantoinase activity; and (b) a C. felis cDNA or a C. felis RNAcomprising a nucleic acid sequence fully complementary to a nucleic acidmolecule of (a).
 2. A method to produce a protein encoded by an isolatedC. felis cDNA or a C. felis RNA nucleic acid molecule selected from thegroup consisting of a C. felis cDNA and a C. felis RNA that hybridizesto a polynucleotide selected from the group consisting of SEQ ID NO:3and SEQ ID NO:6, under conditions comprising (a) hybridizing in asolution comprising 1×SSC in the absence of helix destabilizingcompounds, at a temperature of about 37° C. and (b) washing in asolution comprising 1×SSC in the absence of helix destabilizingcompounds, at a temperature of about 47.5° C., wherein said isolatednucleic acid molecule encodes a protein having allantoinase activity,said method comprising the steps of (1) culturing a cell transformedwith said isolated nucleic acid molecule encoding said proteinoperatively linked to a transcription control sequence and (2)recovering said encoded protein.
 3. A composition comprising anexcipient and an isolated C. felis cDNA or a C. felis RNA nucleic acidmolecule selected from the group consisting of (a) a C. felis cDNA or aC. felis RNA that hybridizes to a polynucleotide selected from the groupconsisting of SEQ ID NO:3 and SEQ ID NO:6 under conditions comprising(1) hybridizing in a solution comprising 1×SSC in the absence of helixdestabilizing compounds, at a temperature of about 37° C. and (2)washing in a solution comprising 1×SSC in the absence of helixdestabilizing compounds, at a temperature of about 47.5° C., whereinsaid isolated nucleic acid molecule encodes a protein havingallantoinase activity; and (b) a C. felis cDNA or a C. felis RNAcomprising a nucleic acid sequence fully complementary to a nucleic acidmolecule of (a).
 4. The nucleic acid molecule of claim 1, wherein saidnucleic acid molecule is selected from the group consisting of: anucleic acid molecule comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:6 and fragments thereof, wherein said fragment comprises at least 25contiguous nucleotides from a nucleic acid sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, and SEQ IDNO:6.
 5. The nucleic acid molecule of claim 1, wherein said isolatednucleic acid molecule encodes a protein comprising SEQ ID NO:2.
 6. Arecombinant nucleic acid molecule comprising a nucleic acid molecule asset forth in claim 1 operatively linked to a transcription controlsequence.
 7. A recombinant virus comprising a nucleic acid molecule asset forth in claim
 1. 8. A recombinant cell comprising a nucleic acidmolecule as set forth in claim
 1. 9. The method of claim 2, wherein saidisolated nucleic acid molecule encodes a protein having an amino acidsequence SEQ ID NO:2.
 10. The method of claim 2, wherein said isolatednucleic acid molecule is selected from the group consisting of: anucleic acid molecule comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NO:1 and SEQ ID NO:4 and a nucleic acidmolecule comprising a fragment of a nucleic acid molecule selected fromthe group consisting of SEQ ID NO:1 and SEQ ID NO:4.
 11. The compositionof claim 3, wherein said composition further comprises a componentselected from the group consisting of an adjuvant and a carrier.
 12. Thecomposition of claim 3, wherein said isolated nucleic acid moleculeencodes a protein having an amino acid sequence SEQ ID NO:2.
 13. Thecomposition of claim 3, wherein said isolated nucleic acid molecule isselected from the group consisting of: a nucleic acid moleculecomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NO:1 and SEQ ID NO:4 and a nucleic acid molecule comprising afragment of a nucleic acid molecule selected from the group consistingof SEQ ID NO:1 and SEQ ID NO:4.